NDUFV1 attenuates renal ischemia–reperfusion injury by improving mitochondrial homeostasis

Abstract Impaired mitochondrial function and dysregulated energy metabolism have been shown to be involved in the pathological progression of kidney diseases such as acute kidney injury (AKI) and diabetic nephropathy. Hence, improving mitochondrial function is a promising strategy for treating renal dysfunction. NADH: ubiquinone oxidoreductase core subunit V1 (NDUFV1) is an important subunit of mitochondrial complex I. In the present study, we found that NDUFV1 was reduced in kidneys of renal ischemia/reperfusion (I/R) mice. Meanwhile, renal I/R induced kidney dysfunction as evidenced by increases in BUN and serum creatinine, severe injury of proximal renal tubules, oxidative stress, and cell apoptosis. All these detrimental outcomes were attenuated by increased expression of NDUFV1 in kidneys. Moreover, knockdown of Ndufv1 aggravated cell insults induced by H2O2 in TCMK‐1 cells, which further confirmed the renoprotective roles of NDUFV1. Mechanistically, NDUFV1 improved the integrity and function of mitochondria, leading to reduced oxidative stress and cell apoptosis. Overall, our data indicate that NDUFV1 has an ability to maintain mitochondrial homeostasis in AKI, suggesting therapies by targeting mitochondria are useful approaches for dealing with mitochondrial dysfunction associated renal diseases such as AKI.

mechanism of AKI damage is the destruction of mitochondrial homeostasis. 9 It has been shown that mitochondrial dysfunction is involved in the pathogenesis of AKI and triggers the progression from AKI towards CKD. [10][11][12][13] Proximal renal tubular dysfunction caused by AKI reduced the activity of mitochondrial complex I and the production of adenosine triphosphate (ATP). 14 In accordance, restoring the function and homeostasis of mitochondria is a feasible strategy to prevent AKI. 15 Therefore, the control of mitochondrial dynamics is likely a useful strategy for dealing with kidney dysfunction induced by ischemia and hypoxia. 16 Complex I (NADH: ubiquinone oxidoreductase) is the first enzyme complex in the mitochondrial respiratory chain, which consists of 45 subunits. 17 Its dysfunction impairs the mitochondrial function in renal tubules during reperfusion, which is often associated with energy deficiency. 18 NDUFV1 is the nuclearencoded structural subunit of complex I. NDUFV1 mutations have been shown to be associated with Leigh syndrome (LS), Leigh-like syndrome (LL), diffuse leukoencephalopathy, and Parkinson's disease. 19 We therefore proposed that NDUFV1 may hold therapeutic potential for treating AKI in ischemia-reperfusion (I/R) model mice.
In the present study, we increased NDUFV1 expression in the kidney to examine the renoprotective roles of NDUFV1 in renal I/R mice. Our results showed that the reinforcement expression of NDUFV1 has multiple benefits against kidney damages induced by renal I/R. For example, NDUFV1 reduces serum creatinine and blood urea nitrogen (BUN), attenuates proximal tubule injury, and mitigates cell apoptosis. All these benefits are likely due to the increased integrity and function of mitochondria, and reduced oxidative stress by NDUFV1. These data indicate that NDUFV1 plays an essential role for the proper functioning of the kidney, and for this reason, NDUFV1 might be a target for treating kidney diseases such as AKI and CKD. week-old male C57BL/6 mice were provided by the Animal Center of Nantong University. Mice were randomly divided into three groups: sham, renal ischemia/reperfusion (I/R) and I/R + NDUFV1 group. The plasmid expressing Ndufv1 was provided by Dr. Cheng Sun. 20 To increase NDUFV1 expression in the kidney, mice received 10 μg of the plasmid expressing Ndufv1 via tail vein injection in 8-12 s. 21 Mice in control group received 10 μg of empty vector in 8-12 s. And 48 h post-plasmid injection, mice were subjected to renal I/R surgery using bilateral renal pedicle ligation as described elsewhere with slight changes. 22 Briefly, after anaesthetization using pentobarbital sodium, renal artery and vein were exposed and blood flow was occluded using a microvascular clamp for 30

| Detection of intracellular reactive oxygen species
And 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Beyotime) was used to detect intracellular reactive oxygen species (ROS). Briefly, DCFH-DA was dissolved in serum-free medium at a concentration of 10 μM and used as working solution. To detect ROS, cells were incubated with DCFH-DA working solution for 20 min at 37°C in dark. After three-time washing in serum-free medium, fluorescence in cells was monitored and captured with a fluorescence microscope.
Fluorescence intensity was quantified by using the ImageJ software.

| Measurements of GSH-PX and SOD activities
Glutathione peroxidase and SOD activities were analysed by using commercial kits form Jiancheng Bioengineering Institute (A005-1-2; A001-3-2) according to the manufacturer's instructions.

| Measurement of complex I activity
Complex I activity was analysed by using a commercial kit (Abcam; ab109721) according to the manufacturer's instructions.

| Measurements of serum creatinine and BUN
A creatinine assay kit and a BUN assay kit (Shanghai Kehua Bioengineering Co., Ltd.) were used for measuring serum creatinine and BUN, respectively. All detections were conducted according to the instructions from the manufacturer.

| Kidney histological examination
Kidney tissues were taken out and immediately fixed in 4% paraformaldehyde/phosphate buffer saline (PBS) and then were embedded in paraffin. The embedded paraffins were cut into 4μm thin slices for Periodic Acid-Schiff (PAS) staining, and the structural changes of the kidney were observed under a light microscope. For renal tubular injury index, the renal tubules in a visual field on PAS staining sections with ×400 magnification were used for semi-quantitative evaluation from 0 to 4 (0, normal interstitium; 1: injured area <25%; 2: injured area from 26% to 50%; 3: injured area from 51% to 75%; 4: injured area >75%). 23

| RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from renal tissues using TRIzol reagent

| Western blot analysis
Kidney tissues were subjected to western blot analysis using a procedure described previously. 24 After blocking nonspecific binding with 5% nonfat milk in phosphate-buffered saline solution or 1 h at room temperature, membranes were incubated Signalling Technology). After three times washing in Tris-buffered saline with Tween (TBST), membranes were then incubated with an appropriate secondary antibody for 1 h at room temperature.
Membranes were developed using a chemiluminescence reagent.
The Image J software was used to analyse the densitometry of the blots.

| Immunostaining
Immunostaining was performed using a procedure described previously. 25 Paraffin-embedded kidney sections (4 μm) were deparaffinized, and ethylene diamine tetra acetic acid (1 mM) was used for antigen retrieval. The slides were incubated with anti-Caspase

| TUNEL staining
Apoptosis in renal tissues was identified by a TUNEL assay with an in-situ Cell Death Detection Kit (11684817910; Roche). The assay was performed according to the manufacturer's instructions.

| Succinic dehydrogenase activity staining
The procedures for succinic dehydrogenase (SDH) activity staining were described elsewhere. 26 Briefly, kidney tissues were cut into 10 μm thick cross sections by using a cryostat-microtome (Leica, CM3050S). The sections were then incubated with SDH working solution containing 20 mM KH 2 PO 4 , 76 mM Na 2 HPO 4 , 5.4% succinic acid disodium salt, 0.02% NBT for 1 h at 37°C. The sections were rinsed three times in PBS and fixed in 10% formalin for 10 min at room temperature. After three time washing with 15% ethanol, images for SDH staining were taken with a microscope.

| Mitochondrial ultra-structure analysis
To observe mitochondrial ultra-structures, a transmission electron microscope was employed to this aim using a previous procedure. 26 Briefly, kidney samples were pre-fixed in cacodylate buffer (0.1 M, pH 7.4), in which paraformaldehyde (2.5%) and glutaraldehyde (2.5%) were included. After that, samples were transferred into osmium tetraoxide (1%) and incubated for 1 h at 4°C. After gradient dehydration in alcohol, samples were oriented longitudinally and embedded in Epon 812, and cut into 70 nm thickness sections, which were then contrasted in lead citrate and uranyl acetate. Finally, these prepared sections were analysed with a transmission electron microscope at 80 kV (JEO Ltd.).

| Statistical analysis
No animals or samples were excluded from the analysis. The data were expressed as mean ± SEM with the results of three independent experiments. One-way analysis of variance (anova) followed by Bonferroni's post hoc test was used for comparison in more than two groups. p < 0.05 was considered statistically significant.

| NDUFV1 attenuates renal injury induced by I/R in mice
To test the potential role of NDUFV1 in the kidney, we increased NDUFV1 expression in mouse kidneys by tail vein injection with a plasmid expressing Ndufv1, and then examined renal function ( Figure 1A). First, we analysed the expression of NDUFV1. As shown in Figure 1B whereas reinforcement of NDUFV1 may play a benefit role against renal I/R-induced damages. In addition, the current strategy using a hydrodynamic-based method can increase exogenous NDUFV1 expression in mouse kidneys. Of note, it has been shown that exogenous gene was predominantly expressed in renal glomeruli and tubules using this gene delivery method. 27 Next, we examined whether NDUFV1 plays a beneficial role in AKI. To this end, blood samples were collected at 36 h post-I/R surgery. In I/R model mice, the renal failure was characterized by significant increases in creatinine and BUN ( Figure 1E,F). However, in the presence of exogenous NDUFV1, these detrimental changes were largely prevented ( Figure 1E,F).
Moreover, we examined the pathological changes of kidney proximal renal tubules by PAS staining. The results showed that the epithelial cells of renal tubules in I/R model mice were flattened, the basement membrane was exposed, and the lumen was dilated (Figure 2A,B).
These damages were alleviated by overexpression of NDUFV1 in kidneys (Figure 2A,B). In summary, increased expression of NDUFV1 in the kidney is a useful strategy for improving renal function in AKI.

| NDUFV1 represses renal apoptosis in I/R model mice
It has been shown that renal cell apoptosis is often associated with the pathological progression of AKI. 28 In accordance, we observed renal tubule epithelial cell apoptosis was increased by renal I/R as evidenced by the increased TUNEL positive cells ( Figure 2C,D).
In the presence of exogenous Ndufv1, the cell apoptosis induced by renal I/R was largely prevented (Figure 2C,D). Furthermore, the expression of cleaved Caspase-3 was dramatically induced by renal I/R; overexpression of NDUFV1 attenuated this trend ( Figure 3A,B). Meanwhile, we analysed the expression of Bax and Bcl-2, two cell apoptosis-related proteins. As shown in Figure 3C, Bax was increased by renal I/R, and this increase was largely mitigated by NDUFV1. On the contrary, Bcl-2 was decreased by renal I/R, NDUFV1 prevented this decline ( Figure 3C,D). As a result, the value for Bax/Bcl-2 was significantly increased in I/R group, which was markedly blunted by NDUFV1 ( Figure 3D). These data clearly indicate that NDUFV1 can reduce renal cell apoptosis induced by renal I/R in mice.

| NDUFV1 improves the integrity of mitochondria in renal I/R model mice
Mitochondrial dysfunction is a common event for renal impairment induced by I/R. 13 We therefore examined mitochondrial function in the following experiments. The observed ultra-structures using a transmission electron microscope (TEM) revealed that many mitochondria in kidneys were damaged by I/R surgery, as evidenced by swelled volume, disrupted crista, and smeared double membrane structure; and these detrimental outcomes were largely attenuated by NDUFV1 ( Figure 4A). The mitochondrial DNA copy number was decreased by renal I/R; overexpression of NDUFV1 prevented this decline ( Figure 4B). The activity of complex I was reduced in kidneys of I/R mice. The forced expression of NDUFV1 partially restored complex I activity ( Figure 4C). The production of ATP in kidneys was reduced by renal I/R; NDUFV1 attenuated this decline ( Figure 4D). Furthermore, we analysed the protein levels of SDHA, HSP60, PHB1, VDHC, and Cox IV, which belong to other electron transport chain complexes. The results showed that all these proteins were decreased by renal I/R; whereas overexpression of NDUFV1 exhibited benefits against these declines ( Figure 4E,F).
Succinate dehydrogenase (SDH) staining showed that NDUFV1 ameliorated the decrease in SDH activity in kidneys ( Figure 5A).
These data indicate that increased expression of NDUFV1 improves the integrity of the mitochondrial electron transport chain complexes in renal I/R mice.

| NDUFV1 mitigates oxidative stress in kidneys induced by renal I/R
Mitochondria have been revealed as a main organelle for producing ROS in cells, which may eventually cause oxidative stress. 29 Therefore, we next examined oxidative stress in kidneys. As shown in Figure 5B, the content of GSH was decreased by renal I/R, whereas NDUFV1 prevented this decline. The increased levels in malondialdehyde (MDA) and protein carbonyl (PC) induced by renal I/R were markedly repressed by NDUFV1 ( Figure 5C). The activities Data are shown as means ± SEM (n = 5). ns: no significance, *p < 0.05, **p < 0.01, ***p < 0.001, by one-way anova.
of glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) were reduced in I/R mouse kidneys. Reinforced expression of NDUFV1 partially rescued these enzyme activities ( Figure 5D).

| Knockdown of Ndufv1 aggravates oxidative stress induced by H 2 O 2 in TCMK-1 cells
The above gain-of-function experiments showed the benefits of NDUFV1 on renal function, next we further examined the renoprotective role of NDUFV1 by loss-of-function studies. To this end, TCMK-1 cells were transfected with siRNAs against Ndufv1 to reduce Ndufv1 expression. Indeed, the mRNA levels of Ndufv1 were decreased by all three tested siRNAs ( Figure 6A). Western blot data further confirmed the knockdown efficiencies of Ndufv1 siRNAs ( Figure 6B,C). Of these siRNAs, siRNA-3 exhibited the best knockdown efficiency and it was chosen for the following experiments.
As shown in Figure 6D, the expression of Cox-IV was decreased by H 2 O 2 ; Ndufv1 knockdown further aggravated this trend. Meanwhile, cleaved Caspase-3 was increased by H 2 O 2 , which was further fortified in Ndufv1 siRNA-3 transfected cells ( Figure 6D,E). The levels of GSH were reduced by H 2 O 2 ; knockdown of Ndufv1 further reduced GSH ( Figure 6F). The increase in MDA induced by H 2 O 2 was strengthened by Ndufv1 siRNA-3 ( Figure 6G). SOD activity was declined in the presence of H 2 O 2 , particularly in Ndufv1 knockdown cells ( Figure 6H). The reduced production of ATP by H 2 O 2 was further declined by Ndufv1 siRNA-3 ( Figure 6I). Moreover, the intracellular production of ROS was increased by H 2 O 2 ; knockdown of Ndufv1 further enhanced this induction ( Figure 6J,K). These data further confirmed the essential role of NDUFV1 in mitochondrial function under oxidative stress.

| DISCUSS ION
In the present study, we found that increased expression of NDUFV1 in the kidney plays a renoprotective role in I/R model mice. For example, NDUFV1 reduces the increased BUN and serum creatinine, attenuates proximal renal tubule injury, and ameliorates cell apoptosis in kidneys. As a member of mitochondrial complex I, NDUFV1 improves mitochondrial function and homeostasis, thus plays a beneficial role against oxidative stress and defected energy supply in renal I/R. These data strongly suggest that NDUFV1, together with other members of complex I, may hold therapeutic potential for treating kidney diseases such as AKI.
In the human body, the kidney is a secondly energy-demanding organ, due to it requires larger amount energy to remove waste from the blood, reabsorb nutrients, regulate the balance of electrolytes and fluid, maintain acid-base balance, and manipulate blood pressure. Therefore, mitochondrial homeostasis is extremely important for the proper functioning of the kidney. It is reasonable to predict that, therefore, maintaining mitochondrial function in the kidney is a involved in several disorders including encephalopathy, myopathy, and Leigh syndrome. [30][31][32] Moreover, NDUFS1 (NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1) is another member of complex I, its mutations also correlated with various diseases such as diabetic cardiomyopathy, myocardial hypertrophy, schizophrenia, leukoencephalopathy, and Leigh syndrome. [33][34][35][36][37] Most recently, deficiency of Ndufs2 in dopaminergic neurons caused Parkinson's disease-like phenotypes in mice. 38 It is worthy to note that, all these diseases mentioned above happen in the brain or heart, both organs are energy-demanding tissues. In the present study, we found that Ndufv1 expression was markedly reduced in kidneys from renal I/R mice. Moreover, knockdown of Ndufv1 in TCMK1-cells further aggravated oxidative stress induced by H 2 O 2 . These evidences implied that complex I is involved in the progression of AKI. In line with this prediction, deficiency in complex I induced by interruption of Ndufs6 induced renal injury including albuminuria and renal fibrosis. 39 Therefore, these evidences strongly suggest that complex I is a promising target for dealing with mitochondrial defects associated disorders including kidney diseases.
In line with the notion mentioned above, reinforcement of complex I seems to be a useful approach for treating mitochondrial dysfunction-related diseases. Indeed, forced expression of Ndufs1 in cardiocytes improves complex I activity and alleviates cardiac dysfunction and myocardial fibrosis. 40 HGC is a newly designed HDAC6 inhibitor, which targets NDUFV1 to attenuate cell injury in dopaminergic neurons treated with neurotoxin MPP + . 20 Furthermore, the recovery of mitochondrial function can accelerate the recovery of podocytes after glomerular injury. 41 In the present study, accordingly, we observed that overexpression of NDUFV1 in the kidney plays a beneficial role in renal I/R mice, as evidenced by decreased BUN and serum creatinine, alleviated proximal renal tubule injury, and reduced cell apoptosis. Our data further confirmed that complex I is a promising therapeutic target for developing drugs against kidney diseases such as AKI. Of note, defects in complex III and IV also have been found to be involved in the pathogenesis of renal dysfunction. 42,43 Therefore, these complexes, as well as complex I, might be therapeutic targets for treating renal failure induced by energy deficiency.
In addition to energy supply, mitochondria also produce ROS as byproducts. Excessive ROS accumulation in the cell may cause detrimental outcomes such as cytochrome c release and caspase activation, and eventually induces cell apoptosis. Indeed, we found that renal I/R induced cleaved caspase-3 expression and cell apoptosis in kidneys.
These detrimental outcomes could be ameliorated by NDUFV1, suggesting NDUFV1 improves the integrity and function of mitochondria.
The TEM and biochemical analyses revealed that the mitochondrial integrity and function in kidneys were improved by NDUFV1.
Furthermore, we observed that increased expression of NDUFV1 plays a beneficial role in the ROS defence system, as evidenced by the increased activities of SOD and GSH-PX in NDUFV1overexpressed kidneys. As we know, SOD converts superoxide anions to hydrogen peroxide (H 2 O 2 ) and oxygen, whereas GSH-PX reduces H 2 O 2 to H 2 O. Both SOD and GSH-PX have shown renoprotective effects in AKI. 44 Therefore, activation of SOD and GSH-PX is likely an alternative way for the observed renoprotective effects of NDUFV1. However, the underlying mechanism remains unclear.
Overall, the present study showed that increased expression of NDUFV1, a member of the mitochondrial complex I, is an effective approach against kidney dysfunction in AKI. At this regard, NDUFV1 plays a pivotal role for improving mitochondrial function and homeostasis in the kidney. By considering the kidney is an energydemanding organ, maintaining mitochondrial integrity, and thus providing sufficient energy are of great importance for the proper functioning of the kidney. Mitochondrial complex I is an entry of proton into electron transport chain to produce ATP, and it plays a pivotal role in energy supply. Therefore, targeting mitochondrial complex I (such as NDUFV1) should be a promising way for dealing with kidney diseases including AKI.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data presented in this study are available from the corresponding author upon reasonable request.