HIF‐1α ameliorates tubular injury in diabetic nephropathy via HO‐1–mediated control of mitochondrial dynamics

Abstract Objectives In diabetic nephropathy (DN), hypoxia‐inducible factor‐1α (HIF‐1α) activation in tubular cells plays an important protective role against kidney injury. The effects may occur via the target genes of HIF‐1α, such as haem oxygenase‐1 (HO‐1), but the exact mechanisms are incompletely understood. Materials and methods Mice with proximal tubule‐specific knockout of HIF‐1α (PT‐HIF‐1α−/− mice) were generated, and diabetes was induced in these mice by streptozotocin (STZ) injection. In addition, to mimic a hypoxic state, cobaltous chloride (CoCl2) was applied to HK‐2 cells. Results Our study first verified that conditional knockout of HIF‐1α worsened tubular injury in DN; additionally, aggravated kidney dysfunction, renal histopathological alterations, mitochondrial fragmentation, ROS accumulation and apoptosis were observed in diabetic PT‐HIF‐1α−/− mice. In vitro study showed that compared to control group, HK‐2 cells cultured under hypoxic ambiance displayed increased mitochondrial fragmentation, ROS production, mitochondrial membrane potential loss and apoptosis. These increases were reversed by overexpression of HIF‐1α or treatment with a HO‐1 agonist. Importantly, cotreatment with a HIF‐1α inhibitor and a HO‐1 agonist rescued the HK‐2 cells from the negative impacts of the HIF‐1α inhibitor. Conclusions These data revealed that HIF‐1α exerted a protective effect against tubular injury in DN, which could be mediated via modulation of mitochondrial dynamics through HO‐1 upregulation.

It is known that hypoxia-inducible factor-1 (HIF-1) is a critical molecule for mitigating hypoxia-induced damage and exists as a heterodimer comprising two subunits: a variable α-subunit and a constitutively expressed β-subunit. Moreover, the α-subunit is typically rapidly degraded by the proteasome in normoxia and is stabilized in hypoxia. 6,7 HIF-1 can perform its transcriptional function only subunits are complexed. [6][7][8] Previous studies have shown that an oxygen deficit is present in DN and that enhancing HIF-1 signalling ameliorates the progression of DN. 3,[9][10][11] Although HIF-1α likely plays a renoprotective role, its precise mechanisms in DN have not yet been fully elucidated.
Unsurprisingly, HIF-1α has a connection with mitochondria because both are related to oxygen metabolism. It has been reported that HIF-1α can stimulate the expression of genes encoding proteins involved in the mitochondrial tricarboxylic acid (TCA) cycle 12 and autophagy 13 regulation to improve mitochondrial morphology and function. 14,15 Previous studies by our laboratory showed the importance and characteristics of mitochondrial dynamics in DN, 16,17 and our results are consistent with those from other studies. 16,18,19 More intriguingly, haem oxygenase-1 (HO-1), a target gene of HIF-1α, [20][21][22] has been reported to restrain hypoxia-induced mitochondrial fission. 23 Collectively, these findings highlight the possibility that HIF-1α impacts mitochondrial function and morphology in kidneys of DN. However, the exact molecular mechanism by which HIF-1α protects against mitochondrial dysfunction in tubular cells in the setting of DN is unknown.
In the present study, mice with proximal tubular cell-specific genetic ablation of HIF-1α were generated, and mice were induced to diabetes by streptozotocin (STZ) injection. In addition, a hypoxic cell model was established by treatment with cobaltous chloride (CoCl 2 ), and these cells were then cultured and subjected to various treatments to investigate related mechanisms, aiming to exploring new therapeutic targets for DN.

| Generation of mice with conditional knockout of HIF-1α
HIF-1α-floxed mice were purchased from the Jackson Laboratory.

| Design in the animal experiment
Nine-week-old male PT-HIF-1α −/− mice and littermate fl/fl mice were randomly divided into four groups: fl/fl mice, PT-HIF-1α −/− mice, diabetic fl/fl mice and diabetic PT-HIF-1α −/− mice. These mice were fasted overnight and then intraperitoneally injected with STZ (50 mg/kg body wt; Sigma-Aldrich) consecutively for 5 days as previously described. 25 Three days after the STZ injection, mice with blood glucose levels of >16.7 mmol/L were selected as diabetic mice for the experiment, and the mice were euthanized after 12 weeks. All mice were monitored by the Animal Care and Use Committee of Central South University (China), and all experiments were performed in precise accordance with the established regulations.

| Assessment of metabolic and physiological parameters
The body weight and blood glucose level were measured biweekly, and blood and urine were collected before euthanasia. The blood glucose level and blood samples were assessed as described previously. 17 Urine N-acetyl-β-d glucosaminidase (NAG) was assessed using an automated colorimetric method (Pacific). Urinary creatinine and albumin were measured with a creatinine assay kit and an Albuwell M kit (Exocell). 24

| Histopathological analysis of kidney
Kidney tissue was excised, cut, fixed with paraformaldehyde and embedded in paraffin. Then, these kidney tissue blocks were sliced into four micrometre thick sections. To evaluate the kidney tissue damage, the sections were stained with haematoxylin-eosin (HE) and periodic acid-Schiff (PAS), 26 and these changes were scored with a semiquantitative scoring system (0-3). 27

| Immunohistochemical (IHC) staining
For IHC studies, paraffin-embedded kidney sections were deparaffinized and hydrated using slide warmers and alcohol. After antigen retrieval, the sections were permeabilized with 3% H 2 O 2 and blocked with 5% bovine serum albumin (BSA). Then, the sections were individ-

| Immunofluorescence (IF) staining
The above-described paraffin-embedded kidney sections were also used for IF staining according to previously published procedures. 26 Briefly, the sections were successively labelled with an anti-HIF-1α primary antibody (ab179483) and the corresponding secondary antibody.

| Establishment of a hypoxic cell model and treatment of cells
A human proximal tubular cell line (HK-2) was acquired from ATCC, and cells were cultured in medium as described previously. 26 To establish the hypoxic cell model, the cells were treated with 300 mmol/L CoCl 2 for 24 hours to 28

| Examination of mitochondrial morphological changes by electron microscopy (EM) and fluorescence staining
Mitochondria in renal tubules were observed by EM as previously described. 17 HK-2 cells were stained with MitoTracker Red (Life Technologies) as previously described. 17

| Analysis of apoptosis and reactive oxygen species (ROS) production
Paraffin-embedded sections were subjected to terminal deoxynucleotidyl transferase dUTP nick end-labelling (TUNEL) following the manufacturer's instructions. 29 A dihydroethidium (DHE) (Invitrogen) probe and MitoSox Red (Invitrogen) were used to evaluate intracellular ROS accumulation in renal tubular tissues and HK-2 cells, respectively.

| Western blot analysis
Renal tissue and HK-2 cells were collected, and total protein was extracted. In addition, mitochondrial and cytoplasmic proteins from HK-2 cells were isolated using a Cell Mitochondria Isolation Kit according to the manufacturer's instructions. 26 Primary proximal tubular epithelial cells were isolated from mice (Method S1), and total protein was extracted. A total of 30 µg of protein were loaded onto an 8%-12% gel and separated via sodium dodecyl sulphate (SDS)polyacrylamide gel electrophoresis (PAGE). Then, proteins were transferred to a membrane and incubated with the following primary antibodies at the appropriate concentrations: antibodies against HIF-1α (ab2185) and HO-1 (ab13248); antibodies against Mfn1

| Mitochondrial membrane potential (ΔΨm) assay
The mitochondrial membrane potential was evaluated with a specific dye. In brief, HK-2 cells were stained with tetramethylrhodamine (TMRE, Molecular Probes) at a final concentration of 1 µmol/L for 30 minutes and were then visualized with confocal microscopy.

| Statistical analyses
The statistical significance of the difference between two groups was evaluated by an unpaired t test in animal and cell studies, and the significance of the difference was denoted by the P value (P < .05, P < .005, P < .001). All statistical analyses were implemented in Graph Pad Prism 8.  Figure 1H). As shown in Figure 1I, the scores of evaluation of tubular and glomerular damage in diabetic PT-HIF-1α −/− mice were the highest. Moreover, the DHE probe and TUNEL assay revealed notable increases in ROS production and apoptosis, respectively, in diabetic PT-HIF-1α −/− mice compared to diabetic fl/fl mice ( Figure 1J,K). In addition, we found greatly increased expression of cleaved caspase-9 and caspase-3 in diabetic PT-HIF-1α −/− mice compared with diabetic fl/fl mice ( Figure 1L,M).  (Figure 2A,B). IHC staining showed that HO-1 expression was substantially increased in diabetic mice compared to non-diabetic mice and was clearly reduced in diabetic PT-HIF-1α −/− mice compared to diabetic fl/fl mice ( Figure 2C, D).   Figure S1. Moreover, hypoxia induced proapoptotic proteins expression, such as cleaved caspase-9 and caspase-3 ( Figure 3G,H).

| Disruption of HIF-1α exacerbates hypoxiainduced mitochondrial damage in HK-2 cells
To explore the effects of HIF-1α, HK-2 cells were cultured in hypoxia and treated with the HIF-1α inhibitor KC7F2 or transfected with a HIF-1α plasmid. As shown in Figure

| Lack of HIF-1α aggravates mitochondrial dysfunction in HK-2 cells exposed to hypoxia through HO-1
Cellular IF staining showed that HO-1 agonist hemin further increased HO-1 expression compared to that in hypoxia and that KC7F2 treatment could not block the effect of hemin ( Figure 5A).

| D ISCUSS I ON
This study demonstrated that HIF-1α improved mitochondrial dysfunction and restricted mitochondria-dependent apoptosis in tubular cells of DN via the HO-1 pathway ( Figure 6). This study provided the first demonstration of the protective role of HIF-1α in tubular cell injury in mice with STZ-induced DN. Moreover, a novel mechanism was proposed wherein the HIF-1α/HO-1 pathway is the pivotal pathway mediating tubular cell mitochondrial dynamics in

DN.
Many studies have demonstrated that hypoxia occurs when there is an imbalance between oxygen supply and consumption, and this imbalance is deemed the major driver of DN. 1,3 In view of this information, we attempted to define the relationship between hypoxia and kidney damage in DN. Semenza et al 7 were the first to F I G U R E 4 Effects of HIF-1α on hypoxia-induced mitochondrial damage in HK-2 cells. A, Representative Western blot bands indicating HIF-1α expression in HK-2 cells treated with KC7F2 (a HIF-1α inhibitor) or transfected with a HIF-1α plasmid in hypoxia. B, Representative mitochondrial morphology, ROS generation and mitochondrial membrane potential as detected by MitoTracker Red (upper), MitoSox Red (middle) and TMRE (below) staining, respectively. C-E, Histograms depicting mitochondrial fragmentation (C), the ROS production (D) and mitochondrial membrane potential (E). F and G, Representative Western blot bands (F) and relative densities (G) of Mfn1, Mfn2, Drp1, p-Drp1(S-616) and Fis1 expression in HK-2 cells treated as described in A. H and I, Expression of mito-Cyto.C, mito-Bax, cyto-Cyto.C, cyto-Bax, c-caspase-9 and c-caspase-3 was revealed by Western blotting(H) and related densitometric analysis (I). J, The HO-1 mRNA levels in HK-2 cells were measured by real-time PCR analysis. K and L, The protein expression of HO-1 was assessed by Western blotting (K) and quantitative analysis (L). *P < .05 vs the normoxia group; # P < .05 vs the hypoxia group. n = 3 | 11 of 14 JIANG et Al.
report on HIF-1, and their study expanded on the identify of HIF-1 as a heterodimer that can respond to hypoxia. The investigation of HIF-1α is particularly valuable in kidney disease since HIF-1α is expressed predominantly in tubular cells, and tubules are susceptible to hypoxia. 8,31,32 Furthermore, studies have shown that HIF-1α governs the initial adaptive response to hypoxia. 3,8 Moreover, recent observations have shown that proximal tubular cells are the initiators of and critical therapeutic targets in diabetic kidney disease. [32][33][34][35] To explore the effects of HIF-1α on tubular damage in DN, mice with proximal tubular cell-specific HIF-1α ablation were generated, and these mice were treated with STZ to establish a mouse model of DN.
The absence of HIF-1α in proximal tubular cells aggravated the kidney damage as evidenced by alterations in morphology and function.
These data revealed that HIF-1α plays a protective role in diabetic kidneys, consistent with the observation that HIF-1α deficiency promotes renal injury in DN. 36,37 Interestingly, studies have suggested that HIF-1α can influence mitochondrial morphology and function. 12,14,15 In addition, our previous studies proved that mitochondria play a vital role in DN, especially regarding mitochondrial dynamics. 16,17,29,38 However, whether mitochondria are involved in the protective effect of HIF-1α against tubular injury in DN is still unknown. To explore this possibility, CoCl 2 was applied to HK-2 cells to mimic hypoxia because it is widely used and recognized as a cell model for chemically induced hypoxia; this model is relatively simple, and the conditions are easy to control.
Moreover, treatment with an appropriate concentration of CoCl 2 simulates a hypoxic state, including the induction of HIF-1α expression 39 in cells such as tubular cells. 28 In the present study, we found that the lack of HIF-1α in tubular cells of diabetic kidneys aggravated mitochondrial injury.
This study showed that the expression of HIF-1α was increased in DN kidneys of DN, consistent with the results of other studies. 3,9 A remaining question is why the level of HIF-1α is elevated in diabetic kidneys when it has protective impacts in renal tissue. The increasing expression and activity of HIF-1α is one type of impaired compensatory response. 40 After the level of HIF-1α is increased, its downstream target genes are activated to suppress hypoxia-induced damage to cells and organs, which exerts renoprotective effects. 9,21 In addition, evidence from recent studies indicates that the activity of HIF in tubules is a lack of compensatory response in DN. 9,30 The possible reasons are as follows: first, superoxide (O 2 − ) may reduce HIF-1 activity by inducing α subunit degradation 3,40,41 ; second, hyperglycaemia could suppress HIF-1α responsive transactivation in tubular cells. [42][43][44] In vitro experiments confirmed that the high glucose-stimulated increase in HIF-1α expression was not significant in tubular cells.

F I G U R E 5
Effects of HO-1 on HIF-1α-regulated mitochondrial fragmentation, ROS generation and apoptosis in HK-2 cells. A, Confocal images showing HO-1 expression (upper) and mitochondria (middle) in HK-2 cells in the normoxia, hypoxia, hypoxia + KC7F2, hypoxia + hemin and hypoxia + hemin+KC7F2 groups. Colocalization of HO-1 with mitochondria was assessed (bellow). B, Confocal images of HK-2 cells stained with MitoSox Red and TMRE. C-E, The proportion of fragmented mitochondria (C) and the fluorescence intensity of MitoSox Red (D) and TMRE (E) were quantified and are shown in the histogram. F and G, Western blot analysis showing the protein expression levels of HO-1, Mfn1, Mfn2, Drp1, p-Drp1(S-616) and Fis1 in HK-2 cells treated as indicated (F), and the bands were analysed by densitometry(G). H and I, Western blot analysis of the levels of Bax, Cyt.C, c-caspase-9 and c-caspase-3 (H) and densitometric analysis of the bands (I). *P < .05 vs the normoxia group; # P < .05 vs the hypoxia group; ※ P < .05 vs the KC7F2 treatment group. n = 3 F I G U R E 6 HIF-1α is a transcription factor that regulates HO-1 gene expression, and HO-1 facilitates mitochondrial fusion (Mfn1 and Mfn2) and inhibits mitochondrial fission (Drp1 and Fis1), thus maintaining mitochondrial homeostasis. Under diabetic conditions, kidney tissues are hypoxic, which induces the expression of HIF-1α through an impaired compensatory response. However, tubular-specific deletion of HIF-1α in the kidneys of diabetic mice exacerbated mitochondrial dysfunction and ROS accumulation and then caused tubular damage, thereby leading to diabetic kidney injury Mitochondrial dynamics describes the two features of mitochondria, which manifest as fusion and fission. [45][46][47] In addition, HO-1 may be the intermediary molecule linking HIF-1α and mitochondrial dynamics. HO-1 is a target gene of HIF-1α 22 and exerts protective effects against kidney damage under diabetic conditions. 23 Studies have shown that HO-1 exerts anti-apoptotic, antioxidant, anti-nitrosative and anti-inflammatory effects, 48-50 by reducing the level of the pro-apoptotic protein Bax and proinflammatory/prooxidant protein iNOS, [49][50][51] and increasing the level of the anti-apoptotic protein Bcl-xl and anti-nitrosative protein bilirubin. 49,50,52 Importantly, recent studies have verified that HO-1 activity affects the function and morphology of mitochondria, especially mitochondrial dynamics. [53][54][55] On the basis of these findings, we sought to determine whether HO-1 is the key modulator of HIF-1α-mediated regulation of mitochondrial dynamics in tubular cells. As expected, under hypoxic conditions, HK-2 cells exhibited increased mitochondrial fission, mitochondrial ROS generation and apoptosis, while these effects were further enhanced by HIF-1α inhibitor treatment. While clearly of great importance, in HK-2 cells cotreated with a HIF-1α inhibitor and a HO-1 agonist after exposure to hypoxia the HO-1 agonist rescued the cells from the negative impact of the HIF-1α inhibitor. These results suggest that HIF-1α is the pivotal node upstream of HO-1 expression, identifying a novel pathway through which HIF-1α modulates mitochondrial dynamics via HO-1 in tubular cell injury in DN.
A recent question addresses the specific molecular mechanism by which HO-1 modulates mitochondrial dynamics. Studies speculated that HO-1 can regulate mitochondrial dynamics 53,54,56 and showed that HO-1/CO may mediate mitochondrial dynamics in leukaemia. 55 These results indicate that HO-1-mediated regulation of alterations in mitochondrial dynamics in tubular cells of DN may occur through the HO-1/CO pathway, a possibility that requires further investigation. Iron overload can damage mitochondria, and HO-1 participates in iron metabolism by degrading haem into ferrous iron. 57,58 However, the increase in HO-1 is also accompanied by ferritin upregulation and leads to effluxion of cellular iron. 59,60 Most importantly, the increase in HO-1 causes a decrease in intracellular free iron content and thus mitigates mitochondrial dysfunction.
This study provides a novel perspective on HIF-1α in DN-by not only developing a new research field in diabetic kidney disease but also laying a foundation for the identification of promising therapeutic targets. In summary, HIF-1α plays a vital role in tubular cell injury in DN and is thus a potential therapeutic target.

CO N FLI C T O F I NTE R E S T
No competing interests related to this article are reported.

AUTH O R CO NTR I B UTI O N S
NJ performed the experiments, generated the data and wrote the manuscript. HZ, YCH, SX, LFZ and XFX performed statistical analyses of the data and discussed the results of the manuscript. LL, MY, YX, LW and PG partially edited the manuscript. YL provided technical support for this study. LS is the guarantor of this study, who conceived and designed this study and edited and discussed this manuscript.

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