Human antigen R regulates hypoxia‐induced mitophagy in renal tubular cells through PARKIN/BNIP3L expressions

Abstract Mitochondrial dysfunction contributes to the pathophysiology of acute kidney injury (AKI). Mitophagy selectively degrades damaged mitochondria and thereby regulates cellular homeostasis. RNA‐binding proteins (RBPs) regulate RNA processing at multiple levels and thereby control cellular function. In this study, we aimed to understand the role of human antigen R (HuR) in hypoxia‐induced mitophagy process in the renal tubular cells. Mitophagy marker expressions (PARKIN, p‐PARKIN, PINK1, BNIP3L, BNIP3, LC3) were determined by western blot analysis. Immunofluorescence studies were performed to analyze mitophagosome, mitolysosome, co‐localization of p‐PARKIN/TOMM20 and BNIP3L/TOMM20. HuR‐mediated regulation of PARKIN/BNIP3L expressions was determined by RNA‐immunoprecipitation analysis and RNA stability experiments. Hypoxia induced mitochondrial dysfunction by increased ROS, decline in membrane potential and activated mitophagy through up‐regulated PARKIN, PINK1, BNIP3 and BNIP3L expressions. HuR knockdown studies revealed that HuR regulates hypoxia‐induced mitophagosome and mitolysosome formation. HuR was significantly bound to PARKIN and BNIP3L mRNA under hypoxia and thereby up‐regulated their expressions through mRNA stability. Altogether, our data highlight the importance of HuR in mitophagy regulation through up‐regulating PARKIN/BNIP3L expressions in renal tubular cells.


| INTRODUC TI ON
Mitochondria provide high energy needs to perform pleiotropic functions of the kidney such as metabolism, nutrients' reabsorption, fluid and electrolytes' balance. 1 Mitochondrial dysfunction caused by ischemia-reperfusion injury is one of the most important contributor in AKI pathogenesis. 2 Maintenance of mitochondrial health is pivotal as they are prime source of ROS and apoptotic regulators. 3 Dysfunction causes decreased ATP production and activation of mitochondrial stress responses. Loss of mitochondrial function has been previously reported in experimental AKI models of sepsis and cisplatin-induced nephrotoxicity. [4][5][6][7] Disturbed mitochondrial dynamics, redox status and energetics are all implicated during AKI. 5,[8][9][10] Thus, mitochondria regulate complex cellular signalling of cell survival and death mechanisms. 11 Removal of dysfunctional mitochondria by organelle-specific autophagy termed as mitochondrial autophagy (mitophagy) replenishes with functional mitochondria. 12 Impaired mitophagy mechanisms might promote inflammation and cell death causing AKI progression to chronic kidney disease (CKD). 13 Since mitophagy process maintains functional mitochondria it is important to understand its regulatory mechanisms.
Mitophagy is a protective response against oxidative damage in AKI. 14 During increased oxidative stress, dysfunctional mitochondria with relatively low membrane potential are segregated during mitochondrial fission process. 15 16,17 Renoprotective role of mitophagy has been demonstrated in a hyperglycaemic rabbit model, 18 acid-loaded metabolic acidosis, 19 high-calorie diet-induced injury, 20 ischemia-reperfusion injury, 17 sepsis 21 and kidney fibrosis. 22 Despite abundant knowledge on mitophagy, the regulatory mechanisms involved in the removal of dysfunctional mitochondria remains elusive. For a better therapeutic strategy, understanding the homeostatic regulatory mitophagy mechanisms involved in AKI is paramount.
RNA-binding proteins (RBPs) regulate gene expression through post-transcriptional processing of RNA. RBPs regulate cellular adaptation to stress response by modulating functionally related proteins through RNA regulation. 23 RBPs specifically bind to the A/U rich regions of the target transcripts through RNA-binding domains (RBDs) and regulate its mRNA stability and translation. 24 In addition, these RBPs bound to the AU rich regions at 3'UTRs of RNA transcripts protect from miRNA-mediated translational repression. [25][26][27] HuR is ubiquitously expressed RBP which is mainly involved in post-transcriptional regulation of RNA. HuR belongs to embryonic lethal abnormal vision (ELAV) family of Hu proteins and regulates cellular functions including proliferation, immune regulation, differentiation, senescence, apoptosis and stress responses. 28 The functional importance of RNA-binding proteins in regulating nuclear-encoded mitochondrial protein expression and mitochondrial function has been reviewed elsewhere. 29 We have previously demonstrated that under hypoxia, HuR regulates cellular autophagy and apoptosis in renal cells. 30 However, the importance of RBPs in mitophagy process is not clear. In this study, we focussed on identifying whether HuR might play an important regulatory role in mitophagy.
Our findings show novel evidence that HuR functionally regulates mitophagy under hypoxia-induced stress in renal tubular cells.
HuR-mediated post-transcriptional function up-regulates PARKIN and BNIP3L expressions through mRNA stabilization and thereby regulates hypoxia-induced mitophagy in renal tubular cells.

| Cell culture and treatment
HK-2 cells were cultured in RPMI 1640 medium with 10% FBS and 1% penicillin/streptomycin in a CO 2 incubator at 37°C. The cells were treated under normoxia or 1% hypoxic conditions at indicated time points and harvested for further analysis.

| Confocal imaging
The HK-2 cells or HuR knockdown cells were allowed to attach in coverslips and then exposed to normoxia or hypoxia conditions.

| Mitochondrial superoxide quantification
The mitochondrial superoxide levels were detected using mitochondrial superoxide detection kit (Abcam 19943). The HK-2 cells were allowed for overnight attachment and exposed for normoxia and hypoxia (6 and 16 hours) conditions. Followed by this, mitoROS working solution was added and incubated at 37℃ for 60 minutes. The fluorescence intensity was measured at 540/590 nm using fluorescence reader (Hitachi Spectrofluorometer).

| Mitochondrial membrane potential (Δψm)
The mitochondrial membrane potential assay was performed using JC-1 cell-permeant dye. After the respective treatment schedule of normoxia and hypoxia, cells were treated with JC-1 dye for 30 minutes. The cells were PBS washed, and fluorescence intensity was measured using a fluorescence reader (Hitachi Spectrofluorometer).

| Western blotting
The cells were exposed to normoxia or hypoxia, and the proteins were isolated using RIPA buffer. The SDS-PAGE gel (10%-12%) separated proteins were transferred to PVDF membrane (PerkinElmer, Life Science). The membranes were incubated in the blocking buffer for 1 minute and treated with primary antibodies overnight at 4°C.
The blots were washed with TBST (thrice, 5 minutes each) and incubated with IgG HRP-linked secondary antibodies for 1 hour at room temperature. The membranes were washed with TBST and exposed to enhanced chemiluminescence (ECL) solution for 1-2 minutes, and images were captured in ImageQuant LAS4000 (GE Healthcare).

| HuR-RNA immunoprecipitation
The RNA immunoprecipitation was performed to analyse HuR bound PARKIN and BNIP3L mRNAs. The experiment was performed as per the manufacturer's protocol (Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit; Millipore). The HuR-RNA complexes in the cell lysate were immunoprecipitated using protein G beads-HuR antibody. The purified RNA was analysed for PARKIN and BNIP3L expressions using RT-qPCR analysis.

| mRNA stability
To determine HuR-mediated RNA stability, WT and knockdown HuR cells were treated with actinomycin D (2.5 μg/mL). The isolated RNAs after treatment were analysed for PARKIN and BNIP3L expressions using RT-qPCR analysis and half-life was calculated. 7SL, stable lncRNA was used as a positive control.

| Statistics
Data are expressed as mean values ± SD. The significant differences were calculated using one-way or two-way analysis of variance followed by Dunnett's or Sidak's or Tukey's multiple comparison analysis. The differences were considered statistically significant for P < .05, P < .01, P < .001. NS represents non-significant. The data were analysed using Prism6 (GraphPad Software Inc).

| Hypoxia induces mitochondrial dysfunction and mitophagy proteins (PINK1/PARKIN, BNIP3/ BNIP3L) in renal tubular cells
In order to understand the effect of hypoxia stress on mitochondrial dysfunction, we analysed mitochondrial ROS, mitochondrial membrane potential and mitophagy-related protein expressions in the renal tubular cells. Figure 1A Figure 1H shows that hypoxia increased LC3 mitochondrial translocation under hypoxia leading to mitophagosome formation compared to control cells. These results show that hypoxia induces mitophagy through increased mitophagosome formation in HK-2 cells.

| Human antigen R (HuR) regulates hypoxiainduced mitophagosome formation in renal tubular cells
To identify whether the RNA-binding protein (HuR) regulates hypoxia-induced mitophagy, the HuR expression was analysed under normoxia and hypoxia. Figure 2A,B shows that hypoxia upregulates HuR expression compared to normoxia. Further, HuR translocated from nucleus to cytoplasm under hypoxia ( Figure 2C).
To evaluate the role of HuR on mitophagosome formation, we established knockdown HuR (shHuR) cells ( Figure 2D). Hypoxiainduced LC3II expressions was significantly decreased in the HuR knockdown cells (Figure 2E,F). Immunofluorescence results of LC3 co-localization with TOMM20 show that HuR knockdown cells significantly reduced hypoxic stress-induced mitophagosome formation ( Figure 2G,H).

| HuR regulates mitochondria co-localization with lysosomes
Further, we analysed the involvement of HuR on mitolysosome formation under hypoxia. Figure 3A
To determine whether BNIP3L translocates to mitochondria under hypoxia, co-localization studies were performed in WT and shHuR cells. The immunofluorescence results showed that knockdown HuR significantly reduced BNIP3L co-localization with the mitochondria under hypoxia ( Figure 5C,D). The findings reveal that HuR involves in the mitophagy process by regulating BNIP3L expression.  F I G U R E 5 HuR regulates hypoxiainduced BNIP3L expression. (A, B) Effect of HuR on hypoxia-induced BNIP3 and BNIP3L expressions. The relative density of western blot results shows that shHuR cells significantly downregulated BNIP3L expressions compared to control cells under hypoxia. The results are shown as mean ± SD, n = 3, *P < .05, compared to WT-normoxia; non-significant (NS), # P < .05, compared to WT-hypoxia; two-way ANOVA followed by Sidak's multiple comparisons test. (C) Immunofluorescence studies on BNIP3L translocation to mitochondria (TOMM20) regulated by HuR under hypoxia. BNIP3L-Red; TOMM20 (green); DAPI (Blue). Scale-10 µm, Magnification-1260X (D) Pearson's coefficient for the BNIP3L colocalization with TOMM20. The values are mean ± SD, n = 3, ***P < .001 compared with HK-2 normoxia, ### P < .001 compared with HK-2-Hypoxia, two-way ANOVA followed by Dunnett's multiple comparisons test

| D ISCUSS I ON
The findings of the study show that hypoxia induces mitophagy in the renal tubular cells. Further, the RNA-binding protein (HuR) regulates PARKIN and BNIP3L expressions through post-transcriptional stabilization of mRNA (Figure 7).
Mitochondria homeostasis plays a critical role in the pathogenesis of AKI. Mitochondria under stress undergo changes in mitochondrial membrane potential, mitochondrial biogenesis, mitochondrial fusion-fission mechanisms and mitophagy. 31 Elevated ROS levels and mitochondrial dysfunction has been reported in cisplatin-induced I/R injury 32 and sepsis-associated AKI. 33 Mitochondrial ROS generation and activation of mitophagy have been demonstrated in contrast-induced AKI. 34 The protective role of mitochondrial clearance through mitophagy has been demonstrated in kidney I/R injury. 35 In addition, several studies demonstrate the adverse effects of impaired mitophagy. 36,37 Notably, insufficient mitochondrial autophagy in the hyperglycaemic rats showed severe organ failure of the liver and kidney. 38 In this present study, we show a rise in oxida- The results are shown as mean ± SD, n = 3, non-significant (NS) between IgG under normoxia and hypoxia, ***P < .001 compared to normoxia; two-way ANOVA followed by Sidak's multiple comparisons test. (E-G) Effects of HuR on the mRNA stability of PARKIN and BNIP3L mRNA expressions were determined in the presence of actinomycin D. Stable lncRNA 7SL was used as control and CCHC-type zinc fingers stimulates the translation of mitochondrial metabolic enzymes. 45 The previous study on endoplasmic reticulum (ER) induced stress showed the predominant role of HuR and TIA-1 on the regulation of cytochrome C expression through translation enhancer and repressor function respectively. 46 HuR was found to stabilize COQ7 enzyme and thereby regulates mitochondrial function through CoQ biosynthesis. 47

| CON CLUS ION
In conclusion, hypoxia induces mitophagy through mitophagosome and mitolysosome formation in renal tubular cells. The findings show that RNA-binding protein-HuR regulates mitophagy through RNA stabilization of PARKIN and BNIP3L expressions.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

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