Decreasing REDD1 expression protects against high glucose-induced apoptosis, oxidative stress and inflammatory injury in podocytes through regulation of the AKT/GSK-3β/Nrf2 pathway

Abstract Objective Our goal in this work was to investigate the possible role and mechanism of regulated in development and DNA damage response 1 (REDD1) in mediating high glucose (HG)-induced podocyte injury in vitro. Materials and methods Mouse podocytes were stimulated with HG to establish HG injury model. Protein expression was examined by Western blotting. Cell viability was measured by cell counting kit-8 assay. Cell apoptosis was assessed by annexin V‐FITC/propidium iodide and TUNEL apoptotic assays. Levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were quantified by commercial kits. Concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β were measured by ELISA. Results A marked increase in REDD1 expression was observed in podocytes stimulated with HG. Reduced REDD1 expression strikingly restrained HG-induced increases in apoptosis, oxidative stress, and inflammation response in cultured podocytes. Decreasing REDD1 expression enhanced nuclear factor erythroid 2-related factor 2 (Nrf2) activation in HG-exposed podocytes via regulation of the AKT/glycogen synthase kinase-3 beta (GSK-3β) pathway. Inhibition of AKT or reactivation of GSK-3β prominently abolished Nrf2 activation induced by decreasing REDD1 expression. Pharmacological repression of Nrf2 markedly reversed the protective effects of decreasing REDD1 expression in HG-injured podocytes. Conclusion Our data demonstrate that decreasing REDD1 expression protects cultured podocytes from HG-induced injuries by potentiating Nrf2 signaling through regulation of the AKT/GSK-3β pathway. Our work underscores the potential role of REDD1-mediated podocyte injury during the development of diabetic kidney disease.


Introduction
Diabetic kidney disease, a severe and common complication of diabetic patients, is a primary cause of end-stage renal disease, affecting health and quality of life [1].Due to unhealthy lifestyles, the incidence of diabetes is rapidly increasing in current years.Unfortunately, quite a few diabetic patients will develop diabetic kidney disease [2].However, effective therapeutic options for diabetic kidney disease are still lacking.Podocytes are a major component of the glomerular filtration barrier, and their damage is a common denominator of glomerular diseases, including diabetic kidney disease [3].Under diabetic conditions, persistent stimulation of podocytes with high glucose (HG) causes excessive apoptosis, oxidative stress, and inflammatory response, which contributes to the development of early diabetic kidney disease [4][5][6].However, the underlying mechanisms involved in HG-induced podocyte damage are currently unclear.Therefore, extensive exploration is required to understand the molecular mechanisms underlying HGinduced podocyte damage, which may hold the key to developing effective treatment strategies for diabetic kidney disease.
Regulated in development and DNA damage response 1 (REDD1) is a multifunctional protein that plays a vital role in diverse physiological and pathological contexts [7].REDD1 was initially identified as a hypoxia-inducible protein and is ubiquitously expressed in various tissues [8,9].Subsequent studies have documented that REDD1 expression can be induced by numerous cellular stresses, including heat shock, radiation, DNA damage, inflammatory response, and energy depletion [10][11][12].Suppression of REDD1 attenuates cell apoptosis and prolongs cell survival under adverse conditions [13,14].REDD1 is involved in the regulation of reactive oxygen species (ROS) production and mediates the sensitivity of cells to oxidative stress [9,14].Moreover, decreasing REDD1 expression represses lipopolysaccharide-induced nuclear factor (NF)-jB activation and proinflammatory cytokine expression [15].Notably, increasing evidence has documented that REDD1 is implicated in various pathological conditions, including cancers, neurological disorders, and diabetes [16][17][18].
Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor protein harboring a basic leucine zipper DNA-binding motif, has been acknowledged as an essential player of the cellular cytoprotective response [19].Typically, Nrf2 enters the nucleus to promote the expression of a group of detoxifying and cytoprotective genes via binding the antioxidant response element (ARE) in promoter regions [20].Therefore, activation of Nrf2 enables cell survival under deleterious stress.Multiple factors contribute to the regulation of Nrf2 activation [21].Glycogen synthase kinase-3 beta (GSK-3b), a multifunctional kinase, plays a vital role in mediating Nrf2 activation [22].GSK-3b is able to phosphorylate Nrf2, which leads to Nrf2 degradation and inactivation, thereby acting as a negative modulator of Nrf2 [23].Simultaneously, the kinase AKT can phosphorylate GSK-3b at the Ser9 residue, resulting in GSK-3b inactivation [24].Therefore, AKT can enhance Nrf2 activation via the inhibition of GSK-3b [25].Notably, Nrf2 has been proposed as a key mediator of podocyte injury in diabetic kidney disease [26,27].However, the regulation of Nrf2 during HG-induced podocyte injury has not been well studied.
Recently, several studies have revealed that REDD1 is implicated in numerous diabetic complications [18,28,29].However, whether REDD1 plays a role in diabetic kidney disease is unknown.Our goal in this investigation was to assess the possible role and mechanism of REDD1 in mediating HGinduced podocyte injury in vitro.

Culture of podocytes
An immortalized murine podocyte cell line, MPC5, was obtained from BeNa Culture Collection (Beijing, China).MPC5 cells were cultivated as per the standard protocol of the manufacturer.Briefly, cells were subjected to RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (Procell, Wuhan, China), which were added with 50 U/ml interferon-c to induce cell proliferation at 33 C. Before being utilized for experiment analysis, cells were subjected to medium without interferon-c and cultured for 2 weeks at 37 C to induce cell differentiation.

Exposure of podocytes to HG
The in vitro model of podocyte injury was established according to previously described protocols [30,31].MPC5 cells were exposed to culture medium containing 30 mM glucose (high glucose, HG) and cultivated for 24 h to induce HG podocyte injury.Podocytes subjected to culture medium containing 5 mM glucose (normal glucose, NG) were applied as a control.

Cell transfection and inhibitor treatment
The siRNAs targeting REDD1 (siRNA-1: 5 0 -ACCGGCUUCAGAG UCAUCAAG-3 0 ; siRNA-2: 5 0 -GGCUGUUAAGUUCUGCCAACU-3 0 ; siRNA-3: 5 0 -GCTAAGUACCGGCUUCAGAGU-3 0 ) and control siRNA (5 0 -UUCUCCGAACGUGUCACGU-3 0 ) were synthesized via GenePharma (Shanghai, China).RNAi-Mate Transfection Reagents (GenePharma) were applied to transfect the siRNAs into MPC5 podocytes following the manufacturer's protocol.In brief, cells were seeded into a 24-well plate and cultured overnight.When cell confluence reached 70-80%, transfection was performed.The siRNAs (20 pmol) were diluted in 50 ll of serum-free medium, while RNAi-Mate transfection reagent (1 ll) was diluted in another 50 ll of serum-free medium.The diluted siRNAs and transfection reagent were mixed and incubated for 20 min at room temperature.The mixture was then added to cells and cultured for 6 h.Thereafter, the medium was replaced by normal growth medium and cultured for 48 h before being harvested for further experiments.GSK-3b-S9A vector was purchased from Addgene (Watertown, MA, USA).The Akt inhibitor MK-2206 2HCI and Nrf2 inhibitor ML385 were purchased from Selleck (Shanghai, China).MK-2206 2HCI and ML385 were diluted in vehicle dimethylsulfoxide (DMSO) for use.MK-2206 2HCI was used to inhibit Akt at a final concentration of 5 lM.ML385 was used to inhibit Nrf2 at a final concentration of 5 lM.

Assay for podocyte viability
MPC5 podocytes seeded in 96-well plates were applied to perform the viability assay.After the indicated transfection and HG stimulation, cell counting kit-8 (CCK-8) reagents (Solarbio, Beijing, China) were added to cells at 10 ll per well.After 2 h at 37 C, the optical density at 450 nm was recorded through a microplate spectrophotometer (BioTek Instruments, Beijing, China) to calculate cell viability.

Assay for podocyte apoptosis
For the annexin V-FITC/propidium iodide (PI) apoptotic assay, detected podocytes were washed and suspended into binding buffer, followed by incubation with annexin V-FITC/PI reagents (Beyotime, Shanghai, China).After being incubated for 20 min at room temperature in the dark, cell samples were assessed with flow cytometry to calculate the cell apoptotic rate.The cells in the quadrants of annexin V-FITC þ /PI -and annexin V-FITC þ /PI þ were counted for calculating apoptosis rate.For the TUNEL apoptotic assay, detected podocytes were washed and fixed with immuno-staining fix solution for 30 min.Then, cells were washed and permeabilized with permeabilization solution for 5 min.Afterwards, cells were washed and incubated with TUNEL detection solution (Beyotime) for 60 min at room temperature under dark conditions.
The nuclei were stained by DAPI.Immunofluorescent cells were observed by a fluorescence microscope.Three random fields were chosen and TUNELpositive cells (green) and total nucleus (blue) were counted to calculate the apoptotic rate following the formula: (number of TUNEL-positive cells/number of total nucleus) Â 100%.

Assay for reactive oxygen species (ROS) detection
Detected podocytes were dissociated and suspended into serum-free media containing 10 lmol/l DCFH-DA (Solarbio, Beijing, China).Cells were placed in a cell incubator and cultured for 20 min at 37 C.Then, cells were washed with serum-free media and detected with flow cytometry to measure the degree of fluorescence.

Determination of redox markers
Cultured podocytes were lysed, and the supernatants were collected.The levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in cell lysates were determined via corresponding commercial colorimetric kits (Beyotime, Shanghai, China) as per the protocol of the manufacturer.

ELISA for detecting pro-inflammatory cytokines
Cultured podocytes were centrifuged at 1000 Â g for 20 min to collect the supernatants.The concentration of tumor necrosis factor (TNF)-a, interleukin (IL)-6, and IL-1b in the supernatants were assessed with corresponding commercially available ELISA kits (Elabscience, Wuhan, China) in compliance with the protocol of the manufacturer.

ARE reporter gene assay
The ARE-luc reporter vector containing ARE sequences (5 0 -ACTGAGGGTGACTCAGCAAAATCACTGAGGGTGACTCAGCA-AAATC-3 0 ) was purchased from Beyotime Biotechnology (Shanghai, China).The ARE-luc reporter vector and REDD1 siRNA were cotransfected into MPC5 podocytes, followed by incubation for 48 h.After HG stimulation, cells were harvested and lysed.Luciferase activities were quantified with a Dual Luciferase Reporter Gene Assay Kit (Beyotime, Shanghai, China) using the protocol of the manufacturer.

Statistical analyses
Data were obtained from three independent experiments.Quantitative results were presented as the mean ± standard deviation.Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's post hoc test were applied for difference comparison.A p-value less than 0.05 was defined as a criterion for statistical significance.

REDD1 expression was elevated in HG-stimulated podocytes
We first assessed the effect of HG stimulation on REDD1 expression alteration in cultured MPC5 podocytes.Our results showed that REDD1 mRNA levels were dramatically elevated in HG-stimulated podocytes compared with control podocytes (Figure 1(A)).Furthermore, we also observed marked increases in REDD1 protein levels in HG-stimulated podocytes (Figure 1(B,C)).Our data display that REDD1 is induced by HG in cultured podocytes.

Decreasing REDD1 expression mitigated HG-evoked podocyte apoptosis
Considering that REDD1 expression is upregulated by HG stimulation in podocytes, we next sought to determine the role of REDD1 inhibition in the regulation of HG-evoked HG podocyte injury.Three siRNAs targeting different sites of REDD1 including REDD1 siRNA-1, REDD1 siRNA-2 and REDD1 siRNA-3 were designed.Notably, the REDD1 siRNA-2 exhibited the best efficacy in silencing of REDD1 expression in cultured podocytes (Supplementary Figure 1).Therefore, the REDD1 siRNA-2 was designated as REDD1 siRNA in the subsequent experiments.REDD1 levels were markedly decreased by transfecting REDD1 siRNAs in cultured podocytes with or without HG stimulation (Figure 2(A-C)).We demonstrated that podocyte viability was markedly impaired via HG stimulation (Figure 2(D)).Strikingly, decreasing REDD1 expression attenuated the deleterious effects  of HG stimulation on podocyte viability (Figure 2(D)).Moreover, decreasing REDD1 expression dramatically reduced the apoptosis of podocytes evoked by HG stimulation, as detected via annexin the TUNEL apoptotic assay (Figure 2(E,F)).In addition, data from the annexin V-FITC/PI apoptotic assay confirmed the inhibitory effects of reduced REDD1 expression on HG-evoked podocyte apoptosis (Figure 2(G,H)).Together, these results illustrate that decreasing REDD1 expression mitigates HG-evoked podocyte apoptosis.

Decreasing REDD1 expression attenuated HG-evoked oxidative stress in cultured podocytes
To further evaluate the role of REDD1 in mediating HG podocyte injury, we next investigated the effects of reduced REDD1 expression on HG-evoked oxidative stress in podocytes.HG stimulation resulted in dramatic increases in intracellular ROS levels in podocytes (Figure 3(A,B)).Notably, reduced REDD1 expression markedly weakened HG-induced increases in ROS levels (Figure 3(A,B)).Furthermore, HG-induced increases in MDA levels were also decreased via decreasing REDD1 expression (Figure 3(C)).In addition, decreasing REDD1 expression strikingly upregulated the levels of antioxidant enzymes SOD (Figure 3(D)) and GPx (Figure 3(E)) in HG-stimulated podocytes.These data hint that the decreasing REDD1 expression attenuates HG-evoked oxidative stress in podocytes.

Decreasing REDD1 expression relieved HG-evoked inflammation response in cultured podocytes
To further explore the role of REDD1 in mediating HG podocyte injury, we detected the effect of reduced REDD1 expression on the HG-evoked inflammation response in podocytes.
Our data exhibited that HG stimulation dramatically enhanced the activation of NF-jB (Figure 4(A,B)) and increased the release of proinflammatory cytokines, including TNF-a, IL-6, and IL-1b (Figure 4(C-E)).Strikingly, the decreasing REDD1 expression markedly restrained HG-induced increases in NF-jB activation (Figure 4(A,B)) and TNF-a, IL-6, and IL-1b levels (Figure 4(C,E)).Overall, these data suggest that decreasing REDD1 expression relieves the HG-evoked inflammation response in podocytes.

Decreasing REDD1 expression potentiated Nrf2 signaling in HG-stimulated podocytes
To illustrate the possible mechanism underlying the protective effects of reduced REDD1 expression in HG-stimulated podocytes, we investigated the role of REDD1 in the regulation of Nrf2 signaling.Our results demonstrated that HG stimulation reduced the levels of Nrf2 in the nucleus (Figure 5(A,B)) and decreased the transcriptional activity of Nrf2/ARE (Figure 5(C)).Notably, reduced REDD1 expression markedly upregulated the levels of nuclear Nrf2 (Figure 5(A,B)) and transcriptional activity of Nrf2/ARE (Figure 5(C)) in HG-stimulated podocytes.Moreover, reduced REDD1 expression significantly increased the expression of Nrf2 target genes, including HO-1 and NQO-1 (Figure 5(D-F)), in HG-stimulated podocytes.Therefore, our data indicate that decreasing REDD1 expression enhances the activation of Nrf2 in HG-stimulated podocytes.

Decreasing REDD1 expression enhanced Nrf2 activation via regulation of the AKT/GSK-3b pathway
To further explore the molecular basis of reduced REDD1 expression on regulating Nrf2 activation, we investigated the effect of decreasing REDD1 expression on the AKT/GSK-3b pathway, which is involved in regulating Nrf2 activation.Our data displayed that decreasing REDD1 expression dramatically increased the phosphorylation of AKT and GSK-3b in HGstimulated podocytes (Figure 6(A-C)).These data indicate that decreasing REDD1 expression enhances AKT activation, while decreasing GSK-3b activation in HG-stimulated podocytes.To verify that decreasing REDD1 expression enhances Nrf2 activation via AKT, we measured the effects of AKT inhibition on the Nrf2 activation induced by reduced REDD1 expression.We found that treatment of the AKT inhibitor  markedly deceased the phosphorylation of AKT and GSK-3b induced by reduced REDD1 expression in HG-stimulated podocytes (Figure 6(A-C)).Notably, inhibition of AKT abolished the Nrf2 activation induced by reduced REDD1 expression in HG-stimulated podocytes (Figure 6(D-F)).Next, we further evaluated the effects of GSK-3b reactivation on the Nrf2 activation induced by reduced REDD1 expression.The constitutively active GSK-3b vector (GSK-3b-S9A) was applied to reactivate GSK-3b in REDD1 siRNA-transfected podocytes (Supplementary Figure 2).Intriguingly, transfection of GSK-3b-S9A markedly reversed the promotion effects of reduced REDD1 expression on Nrf2 activation in HG-stimulated podocytes (Figure 6(G-I)).Moreover, AKT inhibition (Figure 7(A-D)) or GSK-3b reactivation (Figure 7(E-H)) markedly diminished the inhibitory effects of reduced REDD1 expression on HGevoked apoptosis and ROS generation.These results confirm  that decreasing REDD1 expression enhances Nrf2 activation in HG-stimulated podocytes via regulation of the AKT/GSK-3b pathway.

Inhibition of Nrf2 reversed the protective effects mediated by decreasing REDD1 expression in HGinjured podocytes
To validate that REDD1 regulates HG-induced podocyte injury via Nrf2, we further investigated the effect of Nrf2 inhibition on the protective effects mediated by reduced REDD1 expression.Nrf2 activation in REDD1-siRNA-transfected podocytes was markedly decreased via treatment with the Nrf2 inhibitor in HG-stimulated podocytes (Figure 8(A-C)).As expected, the inhibitory effects of reduced REDD1 expression on HG-induced apoptosis (Figure 8(D,E)), ROS production (Figure 8(F,G)), and inflammation response (Figure 8(H-J)) were reversed by Nrf2 inhibition.Therefore, these findings confirm that decreasing REDD1 expression protects from HG-induced podocyte injury via Nrf2.

Discussion
The present work has reported the key role of REDD1 in the regulation of HG-induced injuries of podocytes in vitro.Our findings showed a marked upregulation in REDD1 levels in HG-stimulated podocytes.Loss-of-function experiments for REDD1 elucidated that reduced REDD1 expression protected cultured podocytes against HG-evoked apoptosis, oxidative stress, and inflammatory response.Further research revealed that decreasing REDD1 expression enhanced the activation of Nrf2 signaling via regulation of the AKT/GSK-3b pathway, which was responsible for the protective effects of reduced REDD1 expression in HG-injured podocytes (Figure 9).However, decreasing REDD1 expression had no obvious effect in podocytes under normal glucose conditions, indicating that REDD1 specifically mediates podocyte injury under HG conditions.Therefore, our work underscores the vital relevance of the REDD1/AKT/GSK-3b/Nrf2 pathway in mediating HG-evoked injuries of podocytes.
REDD1 plays a role in several diabetic complications.REDD1 expression is elevated in diabetic rats [18].Increased REDD1 is associated with the development of diabetic muscle atrophy in mice [28].Deletion of REDD1 prevents visual dysfunction induced by diabetic retinopathy [32].Moreover, REDD1 expression was increased in response to HG stimulation in cultured retinal cells [29,32,33].In agreement with these studies, our results demonstrated that REDD1 expression was upregulated in cultured podocytes stimulated with HG.Thus, increased REDD1 may be linked to HG-induced injury in podocytes.
REDD1 regulates cell survival and apoptosis under various stressors.The silencing of REDD1 protects cigarette smokeinduced apoptosis of alveolar septal cells [34].Downregulation of REDD1 attenuates serum deprivationinduced apoptosis of nucleus pulposus cells [35].Upregulation of REDD1 facilitates neuronal apoptosis induced by subarachnoid hemorrhage [13].Moreover, downregulation of REDD1 protects neurons or cardiomyocytes from ischemia/reperfusion-induced apoptosis [14,36].These studies suggest that reduced REDD1 expression produces pro-survival effects.Consistently, the results of our work demonstrated that decreasing REDD1 expression increased podocyte viability under HG stimulation and attenuated HG stimulationinduced podocyte apoptosis.Therefore, our study indicates that reduced REDD1 expression protects podocytes from HGinduced apoptosis.
REDD1 acts as a vital regulator of oxidative stress.Downregulation of REDD1 decreases the levels of ROS in fibroblasts and weakens the sensitivity to oxidative stress [9].Downregulation of REDD1 abolishes neuronal oxidative stress under oxygen glucose deprivation/reoxygenation conditions [14].Silencing of REDD1 reduces the production of ROS in hypoxia/reoxygenation-exposed cardiomyocytes [36].Moreover, increased REDD1 expression is associated with ROS generation in the retina of diabetic mice [33].In M€ uller cells, reduced REDD1expression ameliorates oxidative stress in response to HG conditions [37].In this work, reduced REDD1 expression attenuated HG-induced oxidative stress in cultured podocytes.Thus, our work confirms that REDD1 is involved in regulating HG-evoked oxidative stress.
REDD1 plays a key role in mediating the inflammatory response.Overexpression of REDD1 promotes NF-jB activation and alveolar inflammation in alveolar septal cells [34].Downregulation of REDD1 restrained lipopolysaccharideinduced proinflammatory cytokine release is associated with the downregulation of NF-jB activation [15].Overexpression of REDD1 exacerbates endotoxin-induced inflammation and NF-jB activation [12].In this work, we found that decreasing REDD1 expression repressed HG-induced NF-jB activation and proinflammatory cytokine release in cultured podocytes.Therefore, our data confirm that reduced REDD1 expression has an anti-inflammatory action.
REDD1 has been reported as a novel adjustor of Nrf2 signaling.It is reported that decreased REDD1 expression mediates cellular protection against myocardial ischemia/reperfusion injury via Nrf2-eliminated oxidative stress [36].Moreover, a recent study uncovered that REDD1 ablation enhances the stability and activation of Nrf2 by inhibiting GSK-3b [37].Interestingly, our results further elucidated that reduced REDD1 expression inhibits GSK-3b activation by increasing AKT activation, which underlies Nrf2 activation induced by decreasing REDD1 expression.Indeed, REDD1 acts as an endogenous inhibitor of AKT [38,39].Downregulation of REDD1 enhances the phosphorylation of AKT and GSK-3b [33,40].Consistently, our results also demonstrated that decreasing REDD1 expression promoted the phosphorylation of AKT and GSK-3b in HG-stimulated podocytes.Our data indicate that reduced REDD1 expression increases AKT activation, while decreasing GSK-3b activation.Moreover, we found that AKT inhibition or GSK-3b reactivation strikingly abolished the Nrf2 activation induced by decreasing REDD1 expression.Therefore, our work confirms that reduced REDD1 expression enhances Nrf2 activation by inhibiting GSK-3b via increasing AKT activation.Previous studies have documented that the AKT/GSK-3b axis plays an important role in mediating HG podocyte injury [41,42].Moreover, AKT/GSK-3b axis has been reported as a major regulator for Nrf2 activation in podocytes under HG conditions [43,44].In this work, we identified REDD1 as a novel regulator of AKT/GSK-3b/Nrf2 pathway in podocytes under HG conditions.Our findings may reveal a new mechanism for regulating the AKT/GSK-3b/Nrf2 pathway in podocyte injury of diabetic kidney disease.
It is reported that Nrf2 negatively regulates NF-jB in oxidative stress and inflammatory response [45].Numerous studies have documented that activation of Nrf2 is capable of inhibiting the inflammation response induced by HG in podocytes through suppression of NF-jB activation [43,46,47].In this work, we showed that REDD1 inhibition activated the AKT/GSK-3b/Nrf2 pathway while repressed the NF-jB pathway in podocytes under HG conditions.Notably, inhibition of Nrf2 reversed the suppressive effect of REDD1 silencing on NF-jB-mediated inflammatory response.Therefore, REDD1 mediates the NF-jB pathway via the effect on AKT/GSK-3b/Nrf2 pathway.
This work has investigated the role of REDD1 in mediating podocyte injury using a cellular model.Our results elucidated that decreasing REDD1 expression effectively attenuated HGinduced injury of podocytes.However, whether reduced REDD1 expression is able to alleviate podocyte injury in vivo in diabetic kidney disease remains unclear.Therefore, the precise role of REDD1 in mediating podocyte injury requires further study using an animal model with diabetic kidney disease.
Data from the present work illustrate that reduced REDD1 expression protects cultured podocytes against HG-evoked injuries by potentiating Nrf2 signaling through regulation of the AKT/GSK-3b pathway.Our findings highlight that the REDD1/AKT/GSK-3b/Nrf2 pathway may play a key role in podocyte injury in diabetic kidney disease.Our study represents a remarkable advance in understanding of the mechanism in mediating HG injures of podocytes.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1.Effects of HG on REDD1 expression in cultured podocytes.MPC5 podocytes were subjected to media containing HG (30 mM) or NG (5 mM) and cultivated for 24 h before being harvested for detection.(A) Effects of HG stimulation on REDD1 mRNA level were assessed via the RT-qPCR assay.(B, C) Effects of HG stimulation on REDD1 protein levels, examined via western blotting assay.ÃÃ p < 0.01.The histogram bar shown represents mean ± standard deviation.Statistical analysis was performed using Student's t-test.

Figure 2 .
Figure 2. Effects of reduced REDD1 expression on HG-evoked podocyte apoptosis.REDD1 siRNAs or control siRNAs were transfected into MPC5 podocytes for 48 h, before being subjected to HG stimulation.Levels of REDD1 expression in REDD1 siRNA-transfected podocytes were examined via (A) RT-qPCR and (B, C) western blotting assays (b-actin served as the internal control).(D) Effects of decreasing REDD1 expression on HG-induced deleterious effects on podocyte viability.Effects of decreasing REDD1 expression on HG-induced podocyte apoptosis were assessed via (E, F) TUNEL and (G, H) annexin V-FITC/PI apoptotic assays.Scale bar ¼ 100 lm.ÃÃ p < 0.01 and ÃÃÃ p < 0.001.The histogram bar shown represents mean ± standard deviation.Statistical analysis was performed using ANOVA followed by Tukey's post hoc test.

Figure 3 .
Figure 3. Effects of reduced REDD1 expression on HG-evoked oxidative stress in cultured podocytes.(A, B) Effects of decreasing REDD1 expression on ROS levels in HG-stimulated podocytes were measured via DCFH-DA probe staining.Effects of decreasing REDD1 expression on levels of (C) MDA, (D) SOD and (E) GPx in HGstimulated podocytes were assessed via corresponding colorimetric kits.ÃÃ p < 0.01, ÃÃÃ p < 0.001 and ÃÃÃÃ p < 0.0001.The histogram bar shown represents mean-± standard deviation.Statistical analysis was performed using ANOVA followed by Tukey's post hoc test.

Figure 4 .
Figure 4. Effects of reduced REDD1 expression on the HG-evoked inflammation response in cultured podocytes.(A, B) Effects of decreasing REDD1 expression on the NF-jB p65 protein level in the nucleus, determined via the western blotting assay.Lamin B1 served as the loading control for nuclear proteins.Effects of decreasing REDD1 expression on levels of (C) TNF-a, (D) IL-6, and (E) IL-1b, quantified via an ELISA analysis.ÃÃ p < 0.01 and ÃÃÃ p < 0.001.The histogram bar shown represents mean ± standard deviation.Statistical analysis was performed using ANOVA followed by Tukey's post hoc test.

Figure 5 .
Figure 5. Effects of reduced REDD1 expression on Nrf2 signaling in HG-stimulated podocytes.(A, B) Effects of decreasing REDD1 expression on the levels of nuclear Nrf2, determined via the western blotting assay.Lamin B1 served as the loading control for nuclear proteins.(C) Effects of decreasing REDD1 expression on Nrf2/ARE transcriptional activity, monitored via the luciferase reporter gene assay.(D-F) Effects of decreasing REDD1 expression on HO-1 and NQO-1 protein levels, examined via the western blotting assay.ÃÃ p < 0.01 and ÃÃÃ p < 0.001.The histogram bar shown represents mean ± standard deviation.Statistical analysis was performed using ANOVA followed by Tukey's post hoc test.

Figure 6 .
Figure 6.Effects of AKT inhibition or GSK-3b reactivation on the Nrf2 activation induced by reduced REDD1 expression in HG-stimulated podocytes.MPC5 podocytes were transfected with REDD1 siRNAs and cultured for 48 h with or without the AKT inhibitor MK-2206 2HCl before HG stimulation.Effects of AKT inhibition on levels of (A-C) phospho-AKT and GSK-3b, and (D, E) Nrf2 nuclear protein, measured via the western blotting assay.Lamin B1 served as the loading control for nuclear proteins.(F) Effects of AKT inhibition on Nrf2/ARE transcriptional activity, evaluated via the luciferase reporter gene assay.REDD1 siRNAs and GSK-3b-S9A vectors were cotransfected into MPC5 podocytes and cells were cultivated for 48 h before HG stimulation.(G, H) Effects of GSK-3b reactivation on levels of Nrf2 nuclear protein, examined via the western blotting assay.Lamin B1 served as the loading control for nuclear proteins.(I) Effects of GSK-3b reactivation on Nrf2/ARE transcriptional activity, monitored via the luciferase reporter gene assay.ÃÃ p < 0.01 and ÃÃÃ p < 0.001.The histogram bar shown represents mean ± standard deviation.Statistical analysis was performed using ANOVA followed by Tukey's post hoc test.

Figure 7 .
Figure 7. Effects of AKT inhibition or GSK-3b reactivation on the apoptosis and ROS generation in HG-stimulated podocytes transfected with REDD1 siRNA.(A, B) Effects of AKT inhibition on the apoptosis in HG-stimulated podocytes transfected with REDD1 siRNA, assessed via the annexin V-FITC/PI apoptotic assay.(C, D) Effects of AKT inhibition on the ROS generation in HG-stimulated podocytes transfected with REDD1 siRNA, monitored via DCFH-DA probe staining.(E, F) Effects of GSK-3b reactivation on the apoptosis in HG-stimulated podocytes transfected with REDD1 siRNA, evaluated via the annexin V-FITC/PI apoptotic assay.(G, H) Effects of GSK-3b reactivation on the ROS generation in HG-stimulated podocytes transfected with REDD1 siRNA, measured via DCFH-DA probe staining.ÃÃ p < 0.01, ÃÃÃ p < 0.001 and ÃÃÃÃ p < 0.0001.The histogram bar shown represents mean ± standard deviation.Statistical analysis was performed using ANOVA followed by Tukey's post hoc test.

Figure 8 .
Figure 8. Effects of Nrf2 inhibition on the protective effects mediated by reduced REDD1 expression in HG-injured podocytes.MPC5 podocytes were transfected with REDD1 siRNA and cultured for 48 h with or without the Nrf2 inhibitor ML385 before HG simulation.(A, B) Nrf2 nuclear protein was measured via the western blotting assay.Lamin B1 served as the loading control for nuclear proteins.(C) Nrf2/ARE transcriptional activity was monitored via the luciferase reporter gene assay.(D, E) Apoptotic rate of podocytes was evaluated via the annexin V-FITC/PI apoptotic assay.(F, G) ROS levels were detected via DCFH-DA probe staining.Levels of (H) TNF-a, (I) IL-6, and (J) IL-1b were quantified via an ELISA analysis.ÃÃ p < 0.01, ÃÃÃ p < 0.001 and ÃÃÃÃ p < 0.0001.The histogram bar shown represents mean ± standard deviation.Statistical analysis was performed using ANOVA followed by Tukey's post hoc test.

Figure 9 .
Figure 9.A graphical model illustrating the putative mechanism of the REDD1/AKT/GSK-3b/Nrf2 pathway in the protection against HG podocyte injury.Induction of REDD1 by HG results in blockade of Nrf2 nuclear translocation and activation by affecting the AKT/GSK-3b axis.Decreasing REDD1 expression enhances Akt activation and blocks GSK-3b-mediated inhibition of Nrf2, leading to Nrf2 activation and expression of Nrf2 target genes, which protects against HG podocyte injury.