Advanced Oxidation Protein Product Promotes Oxidative Accentuation in Renal Epithelial Cells via the Soluble (Pro)renin Receptor-Mediated Intrarenal Renin-Angiotensin System and Nox4-H2O2 Signaling

Full-length (pro)renin receptor (fPRR), a research hotspot of the renin-angiotensin system (RAS), plays a serious role in kidney injury. However, the relationship between fPRR and advanced oxidation protein product (AOPP) remains largely unexplored. This study was aimed at exploring the effect of fPRR, especially its 28 kDa soluble form called soluble PRR (sPRR), in AOPP-induced oxidative stress in HK-2 cells, a renal proximal tubular epithelial cell line. Incubation of HK-2 cells with 100 μg/ml AOPP resulted in significant upregulation of fPRR expression and caused an approximately fourfold increase in medium sPRR secretion. However, unmodified albumin did not demonstrate the same effects under the same concentration. Treatment of HK-2 cells with the site-1 protease (S1P) inhibitor PF429242 (40 μM) or S1P siRNA significantly inhibited AOPP-induced sPRR generation. fPRR decoy inhibitor PRO20 and PF429242 treatment for 24 h remarkably attenuated the AOPP-induced upregulation of RAS components. Furthermore, PF429242 significantly reduced the AOPP-stimulated expression of NADPH oxidase 4 (Nox4) and H2O2 expression. The use of a small recombinant protein, named sPRR-His, reversed these alterations. In conclusion, these results provided the first demonstration of AOPP-promoted activation of sPRR. Increased renal proximal tubule Nox4-derived H2O2 contributed to the aggravation of oxidative stress. Targeting S1P-derived sPRR is a promising intervention strategy for chronic kidney disease.


Introduction
Chronic kidney disease (CKD) has emerged as a serious public health issue worldwide because of its high prevalence and high mortality associated with CKD progression and end-stage kidney disease; it is an independent risk factor for cardiovascular disease and all-cause mortality development [1,2]. Therefore, continuing to investigate potential treatments to prevent chronic kidney injury is important.
Investigations suggested the critical role of oxidative stress in the pathogenesis, progression, and complications of CKD [3]. The NADPH oxidase (Nox) family is a major source of reactive oxygen species (ROS) [4]. The most extensively studied ROS include hydroxyl radical, superoxide anion, and H 2 O 2 [5]. Among seven Nox families, Nox4 is the most abundant in the human kidney, predominantly in the proximal tubule [6]. The sole function of Nox4 is to produce H 2 O 2 [7].
Advanced oxidation protein product (AOPP), formed mainly by chlorinated oxidants resulting from myeloperoxidase activity, is produced under oxidative conditions and recognized as a novel marker of oxidative damage [8]. Not only are AOPPs products of chronic oxidative stress but also they could trigger oxidative stress and further stimulate ROS generation, leading to an unstoppable positive-feedback loop [9]. AOPPs activate the intrarenal renin-angiotensin system (RAS) through mechanisms involving CD36, protein kinase C alpha, Nox, and nuclear factor-κB signaling, leading to intrinsic renal cells, including endothelial cells, podocytes, and tubular epithelial cells, and excessive production of intracellular superoxide upon stimulation [10][11][12][13]. However, the molecular mechanisms underlying the pharmacological actions of AOPP remain unclear.
This study investigated the role of S1P-derived sPRR in AOPP-elicited oxidative accentuation injury in renal tubule cells and its potential signaling pathways.

Preparation of AOPP-HSA.
Fatty acid-free HSA solution (30 mg/ml) was incubated with 100 mM HOCl (Fluke) at room temperature for 30 min without free amino acids, free carbohydrates, and lipids. Then, the preparation was dialyzed against phosphate buffer solution (PBS, Life Technologies) to remove free hypochlorous acid at 4°C overnight.

Small
Interfering RNA (siRNA). RNA interference was implemented using siRNA to silence endogenous fPRR, S1P, and Nox4. HK-2 cells were grown to 70%-80% confluence in six-well culture plates and transfected with fPRR, S1P, Nox4 siRNAs, or control nontargeting siRNA. After the HK-2 cells were transfected with siRNA for 24 h with HiPerFect Transfection Reagent (Qiagen), the supernatant was removed with siRNA and AOPP was added for a further 24 h. Then, 48 h after transfection, the cells were harvested and subjected to RNA analysis. For Western blotting analysis, the HK-2 cells were transfected with siRNA for 24 h, the Oxidative Medicine and Cellular Longevity    [10]. In brief, an aliquot sample was incubated with synthetic ACE-specific substrate hippuryl-histidyl-leucine. The liberated His-Leu was converted into a fluorescent product by incubating with o-phthaldialdehyde. Then, the fluorescence of samples was detected with a MRX microplate reader (Dynex Technologies) at an emission wavelength of 495 nm upon excitation at 365 nm.

Western
Blotting. Protein extraction and Western blotting analysis were performed as previously described [16]. Cells were washed three times with cold PBS and lysed with RIPA lysis buffer (Beyotime) with added AEBSF (Beyotime) on ice. The homogenized cells were centrifuged for 10 min at 12,000 g at 4°C. Protein samples (40 μg) were mixed with 5× loading buffer (Beyotime Institute of Biotechnology). The mixture was incubated in a metal bath at 100°C for 10 min. Proteins (30 μg) were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose (Amersham Pharmacia Biotech) membranes for Western blotting. After being transferred, nonspecific binding was blocked with 5% nonfat dry milk in 1× Tris-buffered saline-Tween-20 (TBST) at RT for 1 h. Subsequently, the membranes were incubated with primary antibodies (anti-PRR antibody, Sigma; anti-S1P antibody, Abcam; anti-Nox4, Abcam; and anti-β-actin, Beyotime) overnight at 4°C. After incubation, the membranes were washed again three times with TBST at RT (7 min per wash) and incubated with secondary antibodies (goat anti-rabbit, 1 : 2,500 dilution in TBST; goat anti-mouse 1 : 5,000 dilution in TBST; Beijing Zhongshan Jinqiao Biotechnology) for 1 h. Protein bands were visualized using the ECL detection kit (Beyotime). Densitometric analysis of each band was performed on Image-Pro Plus 6.0.
2.9. Quantitative Real-Time Reverse Transcription-PCR (qRT-PCR). RNA protocols and qRT-PCR were carried out as previously described [34]. Gene expression was normalized to GAPDH. The cells were snap frozen in the TRIzol reagent (CWBio). All samples were treated with DNase digestion during RNA purification by using the RNase-Free DNase kit (Kirgen). RNA was converted to cDNA by using the High-Capacity cDNA Reverse Transcription Kit (Qiagen). The sequences of the primers (Shanghai Sangon Company) are listed in Table 1  In the medium, the induced fPRR was cleaved to generate sPRR, as analyzed by qRT-PCR detection of the increased fPRR mRNA levels and Western blotting detection of the augmented sPRR protein expression; sPRR reached the peak at 100 μg/ml AOPP (Figures 1(a) and 1(b)). In addition, AOPP-HSA (100 μg/ml) upregulated fPRR mRNA expression levels in a time-dependent manner (Figure 1(c)). In order to eliminate the interference of unmodified albumin, exposure of HK-2 cells to AOPP-HSA (100 μg/ml) significantly induced fPRR cleavage events, whereas HSA under the same protein concentration has had no effect. As shown in Figure 1(d), AOPP-HSA (100 μg/ml) upregulated the expression of fPRR mRNA levels, whereas HSA (100 μg/ml) had no effect. As shown in Figure 1(e), AOPP-HSA (100 μg/ml) upregulated the expression of sPRR protein expression, whereas HSA (100 μg/ml) had no effect. As shown in Figure 1(f), AOPP-HSA (100 μg/ml) significantly promoted the secretion of sPRR by more than four times in the medium, whereas HSA (100 μg/ml) had no effect.
These results showed that AOPP-induced fPRR cleavage produced sPRR in HK-2 cells.

AOPP Induced Activation of the RAS System, and
Inhibition of fPRR Significantly Inhibited It. Previous literature reported that AOPP upregulated the expression of nearly all members of RAS in cultured proximal tubule epithelial cells [10]. Therefore, the role of fPRR in AOPPinduced RAS activation was examined. Figure 2(a) shows that the cell-medium renin activity significantly increased in the AOPP (100 μg/ml) group, while it was nearly normalized in the AOPP+PRO20 group. The ACE activity in cell lysates was elevated more than 3.5fold in the AOPP group compared with the control group (CTR), and this elevation was significantly repressed in the AOPP+PRO20 group and AOPP+fPRR siRNA group (p < 0:05, Figure 2(b)). Similar trends were obtained by ). The cell culture medium expressing Ang II was significantly higher in the AOPP group than in the CTR group (p < 0:05), and this expression was also blocked by fPRR siRNA treatment (p < 0:05, Figure 2(f)). Western blotting was then performed to test the silencing effect of siRNA on the expression levels of PRR. Figure 2(g) shows reduced fPRR and sPRR protein expression levels following PRR siRNA treatment.
These results suggested that AOPP may lead to proximal tubule cell injury through the fPRR-mediated activation of renal RAS.

AOPP Induced Activation of Renal Oxidative Stress, and
Inhibition of fPRR Significantly Inhibited It. To the best of the authors' knowledge, compelling evidence revealed the close association between increased AOPP and oxidative stress [35]. Therefore, intercellular adhesion molecule 1 (ICAM-1), TNF-α, TBARS, H 2 O 2 , and the markers for oxidative stress were examined in the present study. Cell lysates' ICAM-1 and TNF-α mRNA levels were significantly higher in the AOPP group than in the control group, and this expression was also blocked in the AOPP+PRO20 and AOPP+fPRR siRNA groups (p < 0:05, Figures 3(a) and  3(b)). Similar results were acquired by ELISA analysis of TBARS and H 2 O 2 (p < 0:05, Figures 3(c) and 3(d)).

AOPP Induced fPRR Cleavage to Produce sPRR, Which Is
Mainly Mediated by S1P. As shown in Figures 4(a) and 4(b), furin inhibitor I could inhibit the cleavage of fPRR, but it did  Figure 1A). This finding may explain the reason for furin inhibitors inhibiting the cleavage of fPRR. However, the S1P inhibitor PF429242 significantly inhibited the cleavage of fPRR to produce sPRR, and the contents of sPRR in the medium were significantly reduced by more than 50% compared with those in the AOPP group (p < 0:05).

AOPP Induced Activation of Renal Local RAS and
Oxidative Stress, and Inhibition of S1P-Derived sPRR Significantly Inhibited This Activation. As shown in Figures 5(a), 5(b), and 5(d), medium renin activity, lysate ACE activity, and Ang II were all significantly higher in the AOPP group than in the CTR group (p < 0:05), and expression was blocked by PF429242 or S1P siRNA treatment (p < 0:05). The protein expression after S1P siRNA knockdown was evaluated by Western blotting analysis. Figure 5(c) shows that S1P siRNA effectively reduced the upregulated expression levels of S1P proteins induced by AOPP.
The mRNA expression levels of AT1R and ACE in the AOPP group were upregulated compared with those in the CTR group (p < 0:05), and these increases were blocked by PF429242 or S1P siRNA (p < 0:05, Figures 5(e) and 5(f)).
Cell lysates and medium were collected and assayed for oxidative stress markers H 2 O 2 and TBARS by using ELISA to test whether S1P-derived sPRR influenced the activation of renal oxidative stress induced by AOPP in HK-2 cells. Medium H 2 O 2 and TBARS were higher in the AOPP group than in the CTR group (p < 0:05), and this expression was blocked by PF429242 (p < 0:05, Figures 5(g) and 5(h)).
3.6. AOPP Induced Activation of the Nox4/H 2 O 2 Pathway, and Inhibition of S1P-Derived sPRR Significantly Inhibited It. ROS includes superoxide anion, H 2 O 2 , hydroxyl radical, and so on [36]. The Nox families catalyze the regulated formation of ROS [37]. Among these Noxs, we found that AOPP induced significant upregulation of Nox4 on mRNA levels but Nox1, Nox2, Nox3, and Nox5 showed no significant difference at the mRNA levels compared to the AOPP group in HK-2 cells as assessed by qRT-PCR (Supplementary Figure 2A-E). Nox4 mainly produces H 2 O 2 rather than superoxide anions, while other Nox isoforms present in the cardiovascular system (i.e., Nox1, Nox2, Nox3, and Nox5) produce superoxide anions as their primary products [4]. In other words, the downstream expression product of Nox4 is H 2 O 2 [38].

Discussion
AOPP was discovered by Witko-Sarsat et al. in 1996 in the plasma of patients who developed chronic renal failure [39]. It is one of the specific markers of protein oxidation, and it is related to the damage of oxygen free radicals in the body and oxidative stress reaction [40]. AOPP is a prod-uct of oxidative stress, and it may induce or aggravate oxidative stress response and chronic inflammation [41]. It is an independent predictor of renal injury in patients with CKD [42]. The level of AOPP was positively correlated with the degree of renal function injury [43,44]. In addition, a growing body of evidence suggested that AOPP could promote the progression of CKD and induce the apoptosis of podocytes, damage of renal tubular epithelium, and proliferation and differentiation of renal mesangial cells [12,[45][46][47]. Studies suggested that AOPP could induce the rapid generation of oxygen free radicals' (0 2− ) renal epithelial cell line, leading to the activation of Nox, which is similar to phagocytes and mainly derived from tubular epithelial cells, and

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Oxidative Medicine and Cellular Longevity the increase in ROS production [48]. A previous study well demonstrated that AOPP contributed to the intrarenal RAS activation involvement of CD36-dependent signaling [10]. However, whether AOPP could activate fPRR and the extracellular domain of soluble PRR-sPRR, a new member of RAS, is unclear. In the current study, AOPP induced fPRR to be cleaved by S1P to generate sPRR, which then activated intrarenal RAS. Meanwhile, sPRR mediated oxidative stress response by affecting the Nox4/H 2 O 2 pathway. Moreover, we also found that Nox4 siRNA treatment did not affect AOPP-induced medium sPRR secretion in HK-2 cells. Therefore, we speculated that other pathways irrelevant to oxidative stress should be included in the secretion of sPRR. Indeed, antioxidant treatment was not sufficient to halt the vicious circle of cause and causality in renal injury. The phenomenon was similar to that found in a previous study [49].
In the Heart Outcomes Prevention Evaluation (HOPE) study, vitamin E (an antioxidant) administered daily for four to six years had no beneficial effects on cardiovascular outcomes in patients at high risk for cardiovascular events. RAS plays an important role in the occurrence and progression of nephropathy [50]. In particular, clinical use of ACEI and ARB could effectively control blood pressure and reduce renal injury, such as proteinuria [51]. fPRR is highly expressed in renal tubules, and it has a potential role in renal RAS regulation [52]. Many studies have shown that fPRR could aggravate tissue damage, such as promoting the development of hypertension, diabetic nephropathy, and proteinuria nephropathy, by activating local RAS in tissues and activating the role of proinflammatory and profibrosis factors [34,[53][54][55][56]. The data in the present study showed that fPRR inhibitors PRO20 and fPRR siRNA could significantly inhibit the AOPP-induced expression levels of AGT, ACE, AT1R, and Ang II in HK-2 cells. Therefore, these novel findings may provide a new therapeutic target for early intervention in the treatment of CKD. Conceivably, the AOPP-related oxidative stress and inflammation to activate RAS and Nox4/H 2 O 2 signaling may be a new therapeutic target for renal intervention in CKD.
Evidence from literature suggested that furin or ADAM19 cleaved fPRR to sPRR, but the phenomenon remains controversial [57,58]. Recent studies have well identified that a novel proprotein convertase, called S1P, is the primary protease responsible for cleaving and producing sPRR [16,17]. In the present study, the S1P inhibitor PF429242 significantly reduced the production of sPRR induced by AOPP. Moreover, two furin inhibitors effectively inhibited fPRR cleavage, but they did not affect sPRR secretion. We also found that mRNA levels of PRR were modestly elevated in treatment with furin inhibitor I compared to AOPP treatment. Such interesting phenomenon needs further investigation. We speculated that a novel sPRR that has not yet been detected by Western blotting and ELISA may be present, which could be cleaved by furin. While we did not have direct evidence, we suspected that furin inhibition reduced the production of the novel sPRR, which further elicited the upregulation of PRR mRNA levels. Overall, these findings extended the previous observations and supported the notion that S1P is the major protease respon-sible for the cleavage and production of sPRR. Previous studies showed that sPRR promoted inflammation via the Nox4/NF-κB pathway and upregulated IL-6, IL-8, and VCAM-1 [31]. Another study highlighted the role of sPRR in renal fibrosis, where sPRR was shown to promote fibronectin in HK-2 cells via the activation of the AKT/βcatenin/snail pathway [28]. The present study revealed that AOPP could activate not only renal RAS but also the Nox4/H 2 O 2 pathway. Inhibition of sPRR could not only reduce the expression levels of renin, Ang II, AGT, ACE, and AT1R but also directly regulate the expression of Nox4 and further affect the production of H 2 O 2 in HK-2 cells. Meanwhile, sPRR-His rescued the expression of Nox4 and the production of H 2 O 2 . These results suggested that sPRR could affect the oxidative stress injury of proximal tubule epithelial cells through RAS-dependent and RASindependent pathways.
Oxidative stress is one of the factors that cause chronic vascular disease [59]. As an important subtype of NADPH oxidase, Nox4 is highly expressed in the kidney, and it could catalyze the production of H 2 O 2 [60]. PRR regulates the stimulating activation of α-ENaC by regulating Nox4mediated H 2 O 2 production [61]. In the present study, sPRR-His and the S1P inhibitor PF429242 were used to investigate the role of sPRR in Nox4 regulation by AOPP. The results showed that AOPP-induced Nox4 expression was significantly inhibited after inhibiting sPRR production, while exogenous sPRR-His recombinant protein could upregulate Nox4 expression. Based on these results, sPRR-His may be involved in regulating the expression of Nox4. However, the specific molecular mechanism of Nox4 by sPRR is still missing, which could be explored in future experiments.
Taken together, this study demonstrated that the sPRR in renal tubular epithelial cells plays an important physiological and pathophysiological role in renal oxidative stress and inflammation through activating intrarenal RAS and Nox4/ H 2 O 2 signaling induced by AOPP.

Data Availability
No data were used to support this study.

Conflicts of Interest
The authors declare that they have no conflicts of interest.