Study on the effect of hydrogen sulfide on oxidative stress in renal tubular epithelial cells injured by hypoxia-reoxygenation


 Background: The present study was designed to determine whether exogenous hydrogen sulfide (H 2 S) inhibits oxidative stress in renal tubular epithelial cells under hypoxia-reoxygenation injury. Material and Methods: NRK-52E cells were divided into 4 group, which were Sham group, HR group, NaHS group and DPI group. Using western blotting to detect the protein expression of NOX2 and NOX4 in the process of hypoxia-reoxygenation. Using real-time quantitative PCR to detect the NOX2 and NOX4 mRNA expression in hypoxia-reoxygenation. The fluorescent probe DCFH-DA was used to detect the content of ROS (reactive oxygen species) in cells. The activity of NADPH oxidase was detected by enzyme-linked immunosorbent assay (ELISA). Results: The protein and mRNA expression of NOX2 and NOX4 were significantly increased in the process of hypoxia-reoxygenation, but decreased with NaHS intervention. ROS content and NADPH oxidase activity were all increased in hypoxia-reoxygenation, but decreased after NaHS intervention. Conclusions: H 2 S can reduce ROS production by inhibiting NOX4 and NOX2 during oxidative stress, and also directly inhibits NADPH oxidase activity.


Introduction
Oxidative stress is an important mechanism of (kidney) ischemia-reperfusion injury [1,2]. Hydrogen sulfide has a protective effect on renal ischemia-reperfusion injury.
Such as, promoting ischemia-reperfusion injury's kidney recovery; promoting the recovery of renal tubular epithelial cells in ischemic injured kidneys. It has been reported that hydrogen sulfide has an inhibitory effect on oxidative stress [3]; however, whether hydrogen sulfide inhibits oxidative stress in renal ischemia-3 reperfusion injury is unknown. This study was designed to investigate the inhibitory effect of hydrogen sulfide on oxidative stress in renal tubular epithelial cells under hypoxia-reoxygenation injury.  After that cells were harvested. NaHS group: cells, cultured in DMEM/F12 containing NaHS concentration of 100 µmol/L, were put in the anoxic gaseous environment with 95% N 2 , 1% O 2 , 4% CO 2 at 37˚C for 60 minutes; and then placed in the gaseous environment with 95% air, 5% CO 2 at 37˚C for 90 minutes. After that cells were harvested. DPI group: cells, cultured in DMEM/F12 containing DPI concentration of 1 µmol/L, were put in the anoxic gaseous environment with 95% N 2 , 1% O 2 , 4% CO 2 at 37˚C for 60 minutes; and then placed in the gaseous environment with 95% air, 5% CO 2 at 37˚C for 90 minutes. After that cells were harvested.

Western blotting analysis
Cells, being intervened, were lysed with ice-cold RIPA buffer containing 1% protease inhibitor PMSF. The supernatant was gathered after centrifugation and quantified using a BCA Protein Assay Kit. All lysates were diluted with SDS loading buffer and Repeat three times.

Statistical Analysis
Each experiment was repeated at least three times independently. Statistical analyses were performed by using SPSS 21.0 software. The results are expressed as the mean ± standard error, unless stated otherwise. Statistical analysis was performed via analysis of variance (one-way ANOVA) followed by the LSD-t test for significance. A P-value of less than 0.05 was considered as statistically significant.

Results
NaHS Decreased the Upregulated NOX2 and NOX4 in Hypoxia-reoxygenation.
In the process of hypoxia-reoxygenation, the protein expression of NOX2 and NOX4 8 was significantly increased in HR group, compared with the Sham group. However, in NaHS group, the protein expression of NOX2 and NOX4 was decreased. The protein expression was almost entirely inhibited in DPI group (Fig. 1).
The variational trend of NOX2 and NOX4 mRNA is the same as their protein level.
Expression of HR group's NOX2 and NOX4 mRNA was increased in hypoxiareoxygenation. In NaHS group, the expression NOX2 and NOX4 mRNA was decreased (Fig. 2).

ROS and NADPH Oxidase Were Downregulated under Stimulation of NaHS.
ROS is an important product in the process of hypoxia-reoxygenation. The result showed that ROS were markedly increased in HR group but reduced in NaHS group.
In DPI group ROS were nearly tally inhibited. (Fig. 3) NOX2 and NOX4 are important components of NADPH oxidase [6,7]. As illustrated in Figs. 4, NADPH oxidase activity was increased in HR group and also reduced in NaHS group (Fig. 3).

Discussion
AKI is common (8-16% of hospital admissions [8,9]), serious (fourfold increased hospital mortality [8,10]), and many aspects of its natural history remain uncertain [11,12]. Currently, the main reasons cause of AKI we have known include sepsis, major surgery, congestive heart failure, higher age and also comorbidity burden of patients that increase the risk of AKI include CKD, proteinuria, diabetes, obesity, and the broadening repertoire of medications that either are directly nephrotoxic or 9 may lower the threshold for sustaining AKI [13]. Clinically, about 75% of patients are caused by renal ischemia reperfusion injury (IRI) [14].
Kidney is a highly perfusion organ, which is sensitive to ischemia and ischemia reperfusion injury. Oxidative stress plays an important role in the development of ischemic AKI. Oxidative stress occurs in various harmful stimuli such as ischemiareperfusion and inflammatory diseases, pathogenic microorganisms produce lipopolysaccharides, and the content of free radicals, reactive oxygen species (ROS) and reactive nitrogen (RNS) in the body increases, and the oxidation degree exceeds the scavenging ability of the body, and tissue damage caused by the imbalance of the oxidation system and antioxidant system [15]. Among them, ROS include superoxide anion (O 2 − ), hydroxyl radical (OH − ) and hydrogen peroxide (H 2 O 2 ). RNS include NO, nitrogen dioxide (NO 2 ) and nitrite peroxide (ONOO − ), etc [16]. After oxidative stress in the body, energy metabolism and cell traits will be change, causing lipid peroxidation, protein denaturation, and abnormal gene expression, eventually leading to various diseases and even death in human bodies [17,18].
The level of oxidative stress is determined by the balance between ROS and antioxidant defense system. Nearly 90% ROS is generated in the mitochondria of human body [19,20]. Mitochondria produce ROS (mtROS) as byproducts of the ATP generation by the oxidative phosphorylation (oxphos) [20]. ROS are formed when one electron is transferred to molecular oxygen, forming the superoxide anion (O 2 − ) [21]. Which means ROS are non-radical and radical reactive molecules [21]. O 2 − can be rapidly dismutated into more stable species such as hydrogen peroxide (H 2 O 2 ), or -in the presence of nitric oxide (NO) -it forms the peroxynitrite anion (ONOO − ) [21]. All of these substances can cause a variety of diseases. When ROS production exceeds the antioxidant capacity of the body, oxidative stress and oxidative damage will occur. The increased oxidative stress level after ischemia reperfusion injury is related to the activation of NADPH oxidase, which is the major enzyme that produces ROS during ischemia-reperfusion injury [22].
NADPH oxidases, are multi-subunit complexes, have seven catalytic subunit homologues which are NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2 [23,24,25]. All of them contain a peroxidase-like domain [23,24,25]. NOX4 is highly expressed in the kidney, and NOX2 and other NOX subunits are also meaningfully expressed in the kidney. They are the source of Reactive oxygen species ROS production in the kidney [26]. ROS produced by NADPH oxidase are involved in mediating many signaling pathways in cells and regulating many physiological activities such as cell growth, cell division, differentiation, migration, apoptosis and aging, which are also closely related to the occurrence and development of diseases [26]. Studies have shown that H 2 S has a direct inhibitory effect on NADPH oxidase activity, and micromole H 2 S can reduce the expression of NADPH oxidase subunit gp91phox (also known as NOX2) [27].
Recent studies have found that H 2 S has many physiological functions, such as relaxing smooth muscle, protecting blood vessels by inhibiting vascular remodeling, participating in shock and inflammatory reactions, participating in nerve activities, and regulating neuroendocrine functions, etc [28]. H 2 S is also considered to be the third gaseous signaling molecule after NO and CO in vivo [28]. Studies have shown that H 2 S has a protective effect on renal ischemia-reperfusion injury. In the renal IRI rat model, the expression of CBS/H 2 S system was significantly decreased. However, after administration of exogenous H 2 S donor NaHS, renal tissue injury and Renal function is improved [29].

Conclusion
In this study, we observed the expression of NOX4 and NOX2, the change of ROS content and the activity of NADPH oxidase, in rat renal tubular epithelial cells pretreated with exogenous hydrogen sulfide at the time of occurring oxidative stress during hypoxia-reoxygenation, and compared with DPI. It is found that: 1.
NOX4 and NOX2 expression and ROS content increased significantly, in renal tubular epithelial cells pretreated without H 2 S after oxidative stress. 2. After pretreatment with H 2 S, the expression of NOX4 and NOX2 and the ROS content were slightly higher than those of normal cells, but there was a significant decrease compared with untreated cells, and the change of NADPH oxidase activity was also the same.
In conclusion, H 2 S can reduce ROS production by inhibiting NOX4 and NOX2 during oxidative stress, and also directly inhibits NADPH oxidase activity.

Funding
The present study was funded by Xinjiang The People's Hospital of Wujiaqu (grant no. 2015AG017).

Availability of data and materials
Data sharing is not applicable to this article, as no data sets were generated or analyzed during the current study.

Ethics approval and consent to participate
Not applicable.

Patient consent for publication
Not applicable.

Figure 1
The protein expression of NOX2 and NOX4 in the HR group was significantly increased compa The mRNA expression of NOX2 and NOX4 in the HR group was increased than that in the Sha  Compared with the Sham group, the NADPH oxidase activity in the HR group was increased (