# Numb Promotes Autophagy through p53 Pathway in Acute Kidney Injury Induced by Cisplatin

Authors: Ze Liu; Yong Li; Yongmei He; Junjie Wang
Journal: Analytical Cellular Pathology
 (2022)

---

## Abstract

Acute kidney injury (AKI) is an important public health concern and characterized as tubular death involved in apoptosis and necrosis. Autophagy is rapidly induced in tubules and associates with renal tubular cells homeostasis to have a complex link with tubular death in AKI. Numb is a multifunctional protein and exerts protective role in tubular death in AKI induced by Cisplatin. However, the effect of Numb on tubular autophagy remains to be investigated. In the present study, the protein expression of LC3 and Beclin-1 related to autophagy was analyzed in Cisplatin-induced AKI mice with knocking down Numb. In model of tubular injury induced by Cisplatinin vitro, downregulation of Numb in NRK-52E cells also inhibited the activation of autophagy accompanied with the decreased protein level of p53. Overexpression of Numb in NRK-52E cells activated autophagy with increased LC3 and Beclin-1 expression accompanied with increased protein level of p53. Moreover, autophagy activation following Numb overexpression was suppressed by p53 inhibitor pifithrin-α. These data indicate that Numb promotes p53-mediated activation of tubular autophagy in AKI induced by Cisplatin and therefore may provide important targets for the treatment of AKI.


---

## Body

## 1. Introduction

With high morbidity and mortality, acute kidney injury (AKI) is a health problem worldwide and leads to heavy socioeconomic burdens [1]. Large number of studies indicated that AKI tends to progress to result in chronic kidney disease and even end-stage kidney disease, besides acute consequences [2]. Cisplatin is a platinum drug for tumor chemotherapy [3–5]. Cisplatin chemotherapy often induced nephrotoxicity and one-third of patients treated with Cisplatin develop AKI [6]. At present, there is no treatment to improve survival outcomes or protect the kidney from Cisplatin-induced AKI, despite advances in research. Increasing studies suggested tubular death including apoptosis and necrosis are the main pathogenetic determinants of Cisplatin-induced AKI, especially in proximal tubular cells, as one of the common targets of Cisplatin-induced AKI [7, 8]. Injured proximal tubules induce renal inflammation and vasoconstriction mediated by inflammatory and vasoactive mediators releasing to further exacerbate tubular damage, which manifests as renal dysfunction [9]. Nevertheless, the complicated pathophysiology underlying Cisplatin-induced tubular injury in AKI remains to be clarified.Autophagy is a dynamic process, during which damaged macromolecules and organelles are transported to the lysosome for degrading and recycling. To support antistress responses and energy maintenance in response to the many stresses including hypoxia, starvation along with oxidative injury involved in the pathogenesis of AKI, autophagy could recycle toxic substances into new cellular components. [10]. During the progress of autophagy, autophagosome with a double-membrane structure formed to sequester and subsequently deliver the cellular constituents to the lysosome for degradation [10]. In mammalian cells, the autophagy-related genes (ATG), such as LC3, also known as ATG8 regulate autophagosomes tightly [11, 12]. LC3-II cleaved and lapidated from LC3-I in the cytoplasm could be translocated onto the membrane of autophagosome rapidly. The LC3-II is considered a reliable marker of autophagy activation [13]. As one of the extraordinary proteins related to autophagy, Beclin-1 is important in recruiting other autophagy-related proteins during membrane expansion in preautophagosome [13].Autophagy can be regulated by mTOR and p53 including their targets in mammals, which was reported in numerous studies [14–19]. As an inhibitor of autophagy and a serine/threonine protein kinase, mTOR is involved in regulating cell growth and proliferation as well as motility and survival [20]. P53 shows a controversial effect on autophagy regulation. As reported in some studies, p53 facilitates autophagy through transactivating a large number of genes such as AMPK and TSC2, which are involved in promoting autophagy [17, 21, 22]. Furthermore, results of some other studies showed that p53 regulates the transcription of PINK1, one of the key mitophagy related proteins encoded by which to suppress autophagy [23]. Therefore, further work is required to identify the effect of autophagy and its regulatory mechanism in AKI induced by Cisplatin.Numb, a multifunctional protein expressed in mammalian cells, was originally authenticated to determine intrinsic cells’ fate in Drosophila with the development of the peripheral and central nervous system [24]. Our previous study demonstrated that Numb attenuates kidney injury induced by Cisplatin by suppressing tubular apoptosis and necrosis [7, 8]. As shown in a recent report, Numb promotes autophagic flux via regulating lysosome function in MCF-7 cells [25]. In our preliminary experimental studies, it was found that Numb promoted the activation of mTOR both in vitro and in vivo. Moreover, Numb is involved in preventing p53 degradation by disrupting the MDM2-p53 complex [26].Together, all the findings suggested that Numb played a role in regulating tubular autophagy. In this regard, we explored the Numb function and mechanism in autophagy regulation in AKI modelsin vitro and in vivo induced by Cisplatin in this study, which would identify important targets for the treatment of AKI.


## 2. Methods

### 2.1. Animal Model

BALB/c mice, male, weighing 20–25 g were supplied by the Center of Experimental Animals in Southern Medical University (Guangzhou, China). All animals were kept at 22-24°C and humidity of 50%-60%, on a cycle of 12 h light and 12 h dark, and freely access water and food at the Xiangnan University Animal Center. To make a mode of AKI-induced Cisplatinin vivo, Cisplatin (p4393; Sigma, St. Louis, MO, USA) was injected intraperitoneally in mice with a single dose of 25 mg/kg, which were sacrificed 3 days after injection. The dose of Cisplatin was chosen according to previous studies, the animals treated with which were induced AKI as well as have the lowest mortality [27–30].


### 2.2. Numb-siRNA Treatment in Mice

The male BALB/c mice (20–25 g) were divided into four groups with 5 mice in each group: (1) Control group: the mice were injected with vehicle intraperitoneally, (2) AKI group: the mice were injected with Cisplatin intraperitoneally, (3) NC-siRNA+AKI group: AKI mice receiving negative control siRNA (NC-siRNA), and (4) Numb-siRNA+AKI group: AKI mice receiving Numb-siRNA. According to the previous studies [31, 32], the mice were administered with Numb-siRNA or NC-siRNA. In brief, 5 5 nM Numb-siRNA or NC-siRNA in 0.2 ml DEPC water was administered within 10 seconds into the mice through the tail vein once a day for 4 days, then the mice were treated with Cisplatin or vehicle intraperitoneally and received siRNA injection every other day. All the mice were euthanized on day 3 after Cisplatin administration. All the animal experiments were according to the approval of the Ethics Committee for Animal Experiments of Xiangnan University.Numb-siRNA modified by 2′-O-methyl partially and NC-siRNA were purchased from RiboBio (Guangzhou, China). The siRNA sequence for knocking down Numb expression in the present study was 5′-CAGCCUGUUUAGAGCGUAAdtdt-3′.


### 2.3. Serum Creatinine and BUN Detection as well as Kidney Damage Pathology

Serum BUN and Creatinine levels were detected by an automatic biochemical analyzer (Au480, Beckman Coulter, CA) based on the manufacturer. HE staining was used to explore the kidney damage pathology following the general protocol. The histology score was determined by counting the percentage of damaged tubules as described previously [7].


### 2.4. Cell Culture and Transfection

NRK-52E cells (Rattus norvegicus, cell strain) were cultured as previously described [33]. NRK-52E cells were completely cultured in the medium of DMEM supplemented with 10% FBS (Invitrogen, Carlsbad, CA). For silencing Numb, 60%-80% confluent NRK-52E cells were transfected with scramble siRNA or Numb-siRNA (Gene pharma, Shanghai, China) with transfection reagent of lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the user’s manual. To activate Cisplatin-induced autophagy, 48 h after transfection, 20 μM Cisplatin was added to cells to incubate for 6 h.Specific siRNA targeting Rat Numb (Numb-siRNA) was designed based on the cDNA sequence of Rat Numb and synthesized by GenePharma (Shanghai, China). Scramble siRNA targeted sense sequence targeted to Scramble siRNA is GCGACGAUCUGCCUAAGAUdTdT, and Numb-siRNA is GCACCUGCCCAGUGGAU CCTT.


### 2.5. Adenovirus Construction and Infection

For Numb overexpression, cDNA of mouse Numb driven by a cytomegalovirus (CMV) promoter involved in recombinant adenovirus vector deficient of serotype 5 in E1 and E3 regions and tagged with hemagglutinin (HA) (Ad-Numb) was constructed and packaged by SinoGenoMax Co. Ltd (http://www.sinogenomax.com, Beijing, China). The adenovirus vector only including CMV and HA was used as control (Ad-ctrl). NRK-52E cells were infected with Ad-ctrl or Ad-Numb for 24 h followed by washing to remove adenovirus. After being cultured in fresh medium for 24 h, the cells were then incubated with Cisplatin (20 μM) for 6 h.To explore the role of p53 in autophagy mediated by Numb, the cells were treated with 20μM pifithrin-α (20 μM, Sigma-Aldrich) or vehicle after Ad-Numb infection.


### 2.6. Western Blotting

The lysate was obtained from kidney tissues or cells in lysis buffer (Merck Millipore, GER) on ice lysing for 30 min. After 12,000 rpm centrifugation at 4°C for 30 min, the supernatants were collected and the concentration of total proteins was quantified using the kit of bicinchoninic acid assay (BCA). Proteins with equal amounts were subjected to Western blot assay according to our previous study [8]. The primary antibodies used in the present study were as follows: Numb (cat: 2756, CST, Beverly, MA) (1 : 1000), p53 (cat: sc-126, Santa, USA) (1 : 1000), LC3 (cat: 4108, CST, Beverly, MA) (1 : 1000), Beclin-1 (cat: 3495, CST, Beverly, MA) (1 : 1000), and GAPDH (cat: 2118, CST, Beverly, MA) (1 : 2000). Relative protein expression was quantified by band intensities measurement using Fluorchem 8900 analysis software (α Innotech Corporation, San Leandro, CA, USA).


### 2.7. Immunofluorescence

To assess autophagy flux, cells cultured on coverslips were processed to co-immunofluorescence staining with LC3 and LAMP1. The slides were fixed using 4% paraformaldehyde in PBS for 15 min at room temperature. After being rinsed twice by PBS, the cells on the slides were permeabilized in Triton X-100(0.05% in PBS) for 30s. The slides were rinsed followed by being incubated with anti-LC3 (cat:4108, CST, Beverly, MA) and LAMP1 (# sc-20011, Santa, USA) at 4°C overnight, and then the cells were incubated with the secondary antibodies (1 : 1000) at room temperature for 1 h in the darkness. The slides were then mounted in Fluoroshield Mounting Medium (Abcam). Fluorescent images of cells were taken using a confocal microscopy (Olympus, Tokyo, Japan).


### 2.8. Statistical Analysis

The data were analyzed statistically using SPSS statistical software (IBM SPSS software, version 19.0). The data were described as means ± SD. Five mice in each group were processed for statistical analysis and triplicate experiments were performedin vitro. The significance among three or more groups was analyzed using one-way analysis of variance (ANOVA). The significance was statistically significant when P value was less than 0.05.


## 2.1. Animal Model

BALB/c mice, male, weighing 20–25 g were supplied by the Center of Experimental Animals in Southern Medical University (Guangzhou, China). All animals were kept at 22-24°C and humidity of 50%-60%, on a cycle of 12 h light and 12 h dark, and freely access water and food at the Xiangnan University Animal Center. To make a mode of AKI-induced Cisplatinin vivo, Cisplatin (p4393; Sigma, St. Louis, MO, USA) was injected intraperitoneally in mice with a single dose of 25 mg/kg, which were sacrificed 3 days after injection. The dose of Cisplatin was chosen according to previous studies, the animals treated with which were induced AKI as well as have the lowest mortality [27–30].


## 2.2. Numb-siRNA Treatment in Mice

The male BALB/c mice (20–25 g) were divided into four groups with 5 mice in each group: (1) Control group: the mice were injected with vehicle intraperitoneally, (2) AKI group: the mice were injected with Cisplatin intraperitoneally, (3) NC-siRNA+AKI group: AKI mice receiving negative control siRNA (NC-siRNA), and (4) Numb-siRNA+AKI group: AKI mice receiving Numb-siRNA. According to the previous studies [31, 32], the mice were administered with Numb-siRNA or NC-siRNA. In brief, 5 5 nM Numb-siRNA or NC-siRNA in 0.2 ml DEPC water was administered within 10 seconds into the mice through the tail vein once a day for 4 days, then the mice were treated with Cisplatin or vehicle intraperitoneally and received siRNA injection every other day. All the mice were euthanized on day 3 after Cisplatin administration. All the animal experiments were according to the approval of the Ethics Committee for Animal Experiments of Xiangnan University.Numb-siRNA modified by 2′-O-methyl partially and NC-siRNA were purchased from RiboBio (Guangzhou, China). The siRNA sequence for knocking down Numb expression in the present study was 5′-CAGCCUGUUUAGAGCGUAAdtdt-3′.


## 2.3. Serum Creatinine and BUN Detection as well as Kidney Damage Pathology

Serum BUN and Creatinine levels were detected by an automatic biochemical analyzer (Au480, Beckman Coulter, CA) based on the manufacturer. HE staining was used to explore the kidney damage pathology following the general protocol. The histology score was determined by counting the percentage of damaged tubules as described previously [7].


## 2.4. Cell Culture and Transfection

NRK-52E cells (Rattus norvegicus, cell strain) were cultured as previously described [33]. NRK-52E cells were completely cultured in the medium of DMEM supplemented with 10% FBS (Invitrogen, Carlsbad, CA). For silencing Numb, 60%-80% confluent NRK-52E cells were transfected with scramble siRNA or Numb-siRNA (Gene pharma, Shanghai, China) with transfection reagent of lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the user’s manual. To activate Cisplatin-induced autophagy, 48 h after transfection, 20 μM Cisplatin was added to cells to incubate for 6 h.Specific siRNA targeting Rat Numb (Numb-siRNA) was designed based on the cDNA sequence of Rat Numb and synthesized by GenePharma (Shanghai, China). Scramble siRNA targeted sense sequence targeted to Scramble siRNA is GCGACGAUCUGCCUAAGAUdTdT, and Numb-siRNA is GCACCUGCCCAGUGGAU CCTT.


## 2.5. Adenovirus Construction and Infection

For Numb overexpression, cDNA of mouse Numb driven by a cytomegalovirus (CMV) promoter involved in recombinant adenovirus vector deficient of serotype 5 in E1 and E3 regions and tagged with hemagglutinin (HA) (Ad-Numb) was constructed and packaged by SinoGenoMax Co. Ltd (http://www.sinogenomax.com, Beijing, China). The adenovirus vector only including CMV and HA was used as control (Ad-ctrl). NRK-52E cells were infected with Ad-ctrl or Ad-Numb for 24 h followed by washing to remove adenovirus. After being cultured in fresh medium for 24 h, the cells were then incubated with Cisplatin (20 μM) for 6 h.To explore the role of p53 in autophagy mediated by Numb, the cells were treated with 20μM pifithrin-α (20 μM, Sigma-Aldrich) or vehicle after Ad-Numb infection.


## 2.6. Western Blotting

The lysate was obtained from kidney tissues or cells in lysis buffer (Merck Millipore, GER) on ice lysing for 30 min. After 12,000 rpm centrifugation at 4°C for 30 min, the supernatants were collected and the concentration of total proteins was quantified using the kit of bicinchoninic acid assay (BCA). Proteins with equal amounts were subjected to Western blot assay according to our previous study [8]. The primary antibodies used in the present study were as follows: Numb (cat: 2756, CST, Beverly, MA) (1 : 1000), p53 (cat: sc-126, Santa, USA) (1 : 1000), LC3 (cat: 4108, CST, Beverly, MA) (1 : 1000), Beclin-1 (cat: 3495, CST, Beverly, MA) (1 : 1000), and GAPDH (cat: 2118, CST, Beverly, MA) (1 : 2000). Relative protein expression was quantified by band intensities measurement using Fluorchem 8900 analysis software (α Innotech Corporation, San Leandro, CA, USA).


## 2.7. Immunofluorescence

To assess autophagy flux, cells cultured on coverslips were processed to co-immunofluorescence staining with LC3 and LAMP1. The slides were fixed using 4% paraformaldehyde in PBS for 15 min at room temperature. After being rinsed twice by PBS, the cells on the slides were permeabilized in Triton X-100(0.05% in PBS) for 30s. The slides were rinsed followed by being incubated with anti-LC3 (cat:4108, CST, Beverly, MA) and LAMP1 (# sc-20011, Santa, USA) at 4°C overnight, and then the cells were incubated with the secondary antibodies (1 : 1000) at room temperature for 1 h in the darkness. The slides were then mounted in Fluoroshield Mounting Medium (Abcam). Fluorescent images of cells were taken using a confocal microscopy (Olympus, Tokyo, Japan).


## 2.8. Statistical Analysis

The data were analyzed statistically using SPSS statistical software (IBM SPSS software, version 19.0). The data were described as means ± SD. Five mice in each group were processed for statistical analysis and triplicate experiments were performedin vitro. The significance among three or more groups was analyzed using one-way analysis of variance (ANOVA). The significance was statistically significant when P value was less than 0.05.


## 3. Results

### 3.1. Knockdown of Numb Specifically in Proximal Tubules Inhibits Autophagy in AKI Induced by Cisplatin

As is known, autophagy plays a crucial role in tubular injury in AKI induced by Cisplatin [10, 34]. Numb protects tubular from injury in a mouse model of AKI induced by Cisplatin [7]. To investigate the effect of Numb on autophagy in AKI induced by Cisplatin, we firstly investigated the autophagy and Numb in injured kidneys of AKI mice induced by Cisplatin. As shown in Figures 1(a) and 1(b), results of Western blotting assay indicated that Cisplatin upregulated the protein expression of autophagy-related LC3-II and Beclin-1, as well as Numb, in the injured kidney after Cisplatin administration in the meantime compared with that in the Control group. Renal injury was confirmed by levels of serum creatinine (Figure 1(c)) and BUN (Figure 1(d)), which are increased significantly in Cisplatin-induced mice along with HE staining for kidney pathology revealed that tubular necrosis with ruptured plasma membrane was observed in the kidney of mice treated with Cisplatin (Figures 1(e) and 1(f)).Figure 1
Depletion of Numb inhibits renal autophagy in AKI model induced by Cisplatin. (a) The protein expression of Numb, Beclin-1, and LC3-II in the kidney from mice 3 days after Cisplatin injection was analyzed by Western blotting. (b) Relative expression of Numb, Beclin-1, and LC3-II to GAPDH was represented in graphic. (c) The level of creatinine in serum. (d) Serum BUN was assayed. (e) HE staining of kidney. (f) Histology score was analyzed. Data are presented as means±SD,n =5. ∗P<0.05 versus Control. #P<0.05 versus scramble siRNA-injected mice treated with Cisplatin. (g) The protein expression of Numb, Beclin-1, and LC3-II in the kidney of mice in each group. (h) Relative expression of Numb, Beclin-1, and LC3-II to GAPDH was represented in graphic. Data are shown with means±SD, n =5. ∗P<0.05 versus Control group. #P<0.05 versus scramble siRNA-injected mice with Cisplatin treatment.
(a)(b)(c)(d)(e)(f)(g)(h)To investigate the role of Numb in autophagy further, the renal expression of Numb was knocked downin vivo by injecting Numb-siRNA. As shown in Figures 1(g) and 1(h), knockdown of Numb expression decreased the renal expression of LC3-II and Beclin-1 significantly in the Cisplatin-induced AKI model. These results suggested that specific loss of Numb in proximal tubule inhibited renal autophagy in AKI induced by Cisplatin.


### 3.2. Silencing Numb Inhibited Autophagy in NRK-52E Cells Induced by Cisplatin

To provide direct evidence that links loss of Numb to tubular cell autophagy, NRK-52E cells, epithelial cells from normal rat kidneys, were transfected with NC-siRNA or Numb-siRNA and then treated with Cisplatin(20μM) for 6 h. As showed in Figure 2(a), Numb-siRNA decreased Numb protein expression significantly compared with scramble siRNA(NC-siRNA). Results of Western blotting revealed that autophagy-related protein LC3-II was upregulated in Cisplatin-treated cells compared with the Control group. However, the LC3-II expression was decreased significantly in cells transfected with Numb-siRNA compared with those transfected with scramble siRNA after Cisplatin administration (Figures 2(b) and 2(c)). Cisplatin induces autophagy which would be attenuated by Numb-siRNA indicated by immunofluorescence staining of LC3 and LAMP1(Figures 2(d) and 2(e)). mTOR is the critical molecule in regulating Cisplatin-induced autophagy in tubular cells [14]. We thus hypothesized that Numb promotes autophagy activation via inhibiting mTOR pathway. Therefore, we explored the effect of Numb on the activity of mTOR. Western blotting revealed that loss of Numb specific to proximal tubular cells that led to the activation of mTOR was inhibited in vitro and in vivo in our preliminary experiments. Our results suggested that Numb might promote autophagy through other pathways rather than the mTOR pathway.Figure 2
Silencing Numb inhibits Cisplatin-induced autophagy in NRK-52E cells. NRK-52E cells were transfected with scramble siRNA or Numb-siRNA followed by 20μM Cisplatin or vehicle (saline) for 6 hours. (a) The interference efficiency of Numb was measured by Western blot assay. (b) The protein expression of Numb, p53, and LC3-II in cells of each group was detected by Western blotting; GAPDH was used as loading control. (c) Relative expression of Numb, p53, and LC3-II to GAPDH was represented in graphic. Data are shown with means±SD, n =3. ∗P<0.05 versus cells treated with vehicle. #P<0.05 versus scramble siRNA-transfected cells with Cisplatin treatment. (d) Representative immunofluorescence images of co-expression of LC3-II (green) and LAMP1 (red) in cells of different groups as indicated. Magnified 600 times. (e) Quantitative statistics for immunofluorescence images. Data are shown as means±SD, n =3. ∗∗P<0.01 versus Control group. ##P<0.01 versus scramble siRNA-transfected cells with Cisplatin treatment.
(a)(b)(c)(d)(e)p53 is another critical molecule in the regulation of autophagy; however, its role is controversial [17–19]. Numb inhibits the degradation of p53 via impacting MDM2-p53 complex [26]. Therefore, Numb was hypothesized to stabilize p53 to promote autophagy. The effect of Numb on p53 expression in NRK-52E cells was further investigated in the present study. P53 expression was upregulated significantly in NRK-52E cells administered with Cisplatin compared with the control cells and significantly decreased in cells transfected with Numb-siRNA compared with those transfected with scramble siRNA (Figure 2(b)). Furthermore, the autophagy marker protein LC3-II was also decreased in cells transfected with Numb-siRNA compared with those transfected with scramble siRNA (Figures 2(b) and 2(c)). In brief, all the results suggested Numb-activated autophagy in proximal tubular cells may be mediated by p53 pathway.


### 3.3. Overexpression Numb Promoted Cisplatin-Induced Autophagy in NRK-52E Cells

To further confirm the role of Numb in autophagy induced by Cisplatin in the tubular cells, we infected NRK-52E cells with overexpression HA-tagged Numb adenovirus encoding (pAd-HA-Numb). It was shown in Figure3(a) that Numb expression detected by Western blot was upregulated significantly in NRK-52E cells infected with pAd-HA-Numb compared with pAd-HA infected group. Moreover, the autophagy marker proteins LC3-II and p53 were increased significantly in cells infected with pAd-HA-Numb compared with pAd-HA infected cells after Cisplatin administration (Figures 3(b) and 3(c)). Overexpression of Numb enhanced Cisplatin-induced autophagy, which was confirmed by immunofluorescence detection for LC3 and LAMP1 (Figures 3(d) and 3(e)). These data further confirmed that Numb may activate autophagy through p53 pathway.Figure 3
Overexpression Numb promotes autophagy in NRK-52E cells induced by Cisplatin. NRK-52E cells were infected with Ad-ctrl or Ad-Numb followed by treated with Cisplatin or vehicle (saline) for 6 hours. (a) The overexpression of Numb was confirmed using Western blotting assay.∗P<0.05 versus cells treated with vehicle. (b) The protein expression of Numb, p53, and LC3-II in cells of different groups as indicated, GAPDH was used as loading control. (c) Relative expression of Numb, p53, and LC3-II to GAPDH was represented in graphic. (d) Representative immunofluorescence images of co-expression of LC3-II (green) and LAMP1 (red) in cells of different groups as indicated. Magnified 400 times. (e) Quantitative statistics for immunofluorescence images. Data are shown as means±SD of three independent experiments. ∗P<0.05, ∗∗P<0.01 versus cells treated with saline-treated cells (Control). ∗P<0.05 versus vehicle-treated cells. #P<0.05, ##P<0.01 versus Ad-Ctrl infected cells with Cisplatin treatment. Data are shown as means±SD. n =3. ∗P<0.05 versus cells treated with saline (Control).
(a)(b)(c)(d)(e)


### 3.4. Numb Activates Cisplatin-Induced Autophagy in p53-Dependent Pathway

In terms of mechanism, to explore the role of p53 in autophagy promoted by Numb, NRK-52E cells infected with Ad-Numb were treated with an inhibitor of p53 as pifithrin-α (PIF-α) for 24 h. Western blotting showed that the protein expression of p53 was decreased significantly in PIF-α-treated cells compared with those treated with vector. Correspondingly, the autophagy-related LC3-II protein expression was decreased in PIF-α-treated cells significantly compared with vector-treated cells (Figure 4). These data indicated Numb-activated autophagy induced by Cisplatin in proximal tubular cells via p53-dependent pathway.Figure 4
Numb activates Cisplatin-induced autophagy in p53-dependent pathway. NRK-52E cells were infected with Ad-Numb for 24 h, then incubated with 20μM PIF-α or vehicle (DMSO) for 1 hour and followed by Cisplatin incubation for 6 hours. (a) The protein expression of Numb, p53, and LC3-II in cells of different groups as indicated, GAPDH was used as loading control. (b) Relative expression of Numb, p53 ,and LC3-II to GAPDH was represented in graphic. Data are shown as means±SD. n =3. ∗P<0.05 DMSO-treated Ad-Numb-infected cells with Cisplatin incubation.
(a)(b)


## 3.1. Knockdown of Numb Specifically in Proximal Tubules Inhibits Autophagy in AKI Induced by Cisplatin

As is known, autophagy plays a crucial role in tubular injury in AKI induced by Cisplatin [10, 34]. Numb protects tubular from injury in a mouse model of AKI induced by Cisplatin [7]. To investigate the effect of Numb on autophagy in AKI induced by Cisplatin, we firstly investigated the autophagy and Numb in injured kidneys of AKI mice induced by Cisplatin. As shown in Figures 1(a) and 1(b), results of Western blotting assay indicated that Cisplatin upregulated the protein expression of autophagy-related LC3-II and Beclin-1, as well as Numb, in the injured kidney after Cisplatin administration in the meantime compared with that in the Control group. Renal injury was confirmed by levels of serum creatinine (Figure 1(c)) and BUN (Figure 1(d)), which are increased significantly in Cisplatin-induced mice along with HE staining for kidney pathology revealed that tubular necrosis with ruptured plasma membrane was observed in the kidney of mice treated with Cisplatin (Figures 1(e) and 1(f)).Figure 1
Depletion of Numb inhibits renal autophagy in AKI model induced by Cisplatin. (a) The protein expression of Numb, Beclin-1, and LC3-II in the kidney from mice 3 days after Cisplatin injection was analyzed by Western blotting. (b) Relative expression of Numb, Beclin-1, and LC3-II to GAPDH was represented in graphic. (c) The level of creatinine in serum. (d) Serum BUN was assayed. (e) HE staining of kidney. (f) Histology score was analyzed. Data are presented as means±SD,n =5. ∗P<0.05 versus Control. #P<0.05 versus scramble siRNA-injected mice treated with Cisplatin. (g) The protein expression of Numb, Beclin-1, and LC3-II in the kidney of mice in each group. (h) Relative expression of Numb, Beclin-1, and LC3-II to GAPDH was represented in graphic. Data are shown with means±SD, n =5. ∗P<0.05 versus Control group. #P<0.05 versus scramble siRNA-injected mice with Cisplatin treatment.
(a)(b)(c)(d)(e)(f)(g)(h)To investigate the role of Numb in autophagy further, the renal expression of Numb was knocked downin vivo by injecting Numb-siRNA. As shown in Figures 1(g) and 1(h), knockdown of Numb expression decreased the renal expression of LC3-II and Beclin-1 significantly in the Cisplatin-induced AKI model. These results suggested that specific loss of Numb in proximal tubule inhibited renal autophagy in AKI induced by Cisplatin.


## 3.2. Silencing Numb Inhibited Autophagy in NRK-52E Cells Induced by Cisplatin

To provide direct evidence that links loss of Numb to tubular cell autophagy, NRK-52E cells, epithelial cells from normal rat kidneys, were transfected with NC-siRNA or Numb-siRNA and then treated with Cisplatin(20μM) for 6 h. As showed in Figure 2(a), Numb-siRNA decreased Numb protein expression significantly compared with scramble siRNA(NC-siRNA). Results of Western blotting revealed that autophagy-related protein LC3-II was upregulated in Cisplatin-treated cells compared with the Control group. However, the LC3-II expression was decreased significantly in cells transfected with Numb-siRNA compared with those transfected with scramble siRNA after Cisplatin administration (Figures 2(b) and 2(c)). Cisplatin induces autophagy which would be attenuated by Numb-siRNA indicated by immunofluorescence staining of LC3 and LAMP1(Figures 2(d) and 2(e)). mTOR is the critical molecule in regulating Cisplatin-induced autophagy in tubular cells [14]. We thus hypothesized that Numb promotes autophagy activation via inhibiting mTOR pathway. Therefore, we explored the effect of Numb on the activity of mTOR. Western blotting revealed that loss of Numb specific to proximal tubular cells that led to the activation of mTOR was inhibited in vitro and in vivo in our preliminary experiments. Our results suggested that Numb might promote autophagy through other pathways rather than the mTOR pathway.Figure 2
Silencing Numb inhibits Cisplatin-induced autophagy in NRK-52E cells. NRK-52E cells were transfected with scramble siRNA or Numb-siRNA followed by 20μM Cisplatin or vehicle (saline) for 6 hours. (a) The interference efficiency of Numb was measured by Western blot assay. (b) The protein expression of Numb, p53, and LC3-II in cells of each group was detected by Western blotting; GAPDH was used as loading control. (c) Relative expression of Numb, p53, and LC3-II to GAPDH was represented in graphic. Data are shown with means±SD, n =3. ∗P<0.05 versus cells treated with vehicle. #P<0.05 versus scramble siRNA-transfected cells with Cisplatin treatment. (d) Representative immunofluorescence images of co-expression of LC3-II (green) and LAMP1 (red) in cells of different groups as indicated. Magnified 600 times. (e) Quantitative statistics for immunofluorescence images. Data are shown as means±SD, n =3. ∗∗P<0.01 versus Control group. ##P<0.01 versus scramble siRNA-transfected cells with Cisplatin treatment.
(a)(b)(c)(d)(e)p53 is another critical molecule in the regulation of autophagy; however, its role is controversial [17–19]. Numb inhibits the degradation of p53 via impacting MDM2-p53 complex [26]. Therefore, Numb was hypothesized to stabilize p53 to promote autophagy. The effect of Numb on p53 expression in NRK-52E cells was further investigated in the present study. P53 expression was upregulated significantly in NRK-52E cells administered with Cisplatin compared with the control cells and significantly decreased in cells transfected with Numb-siRNA compared with those transfected with scramble siRNA (Figure 2(b)). Furthermore, the autophagy marker protein LC3-II was also decreased in cells transfected with Numb-siRNA compared with those transfected with scramble siRNA (Figures 2(b) and 2(c)). In brief, all the results suggested Numb-activated autophagy in proximal tubular cells may be mediated by p53 pathway.


## 3.3. Overexpression Numb Promoted Cisplatin-Induced Autophagy in NRK-52E Cells

To further confirm the role of Numb in autophagy induced by Cisplatin in the tubular cells, we infected NRK-52E cells with overexpression HA-tagged Numb adenovirus encoding (pAd-HA-Numb). It was shown in Figure3(a) that Numb expression detected by Western blot was upregulated significantly in NRK-52E cells infected with pAd-HA-Numb compared with pAd-HA infected group. Moreover, the autophagy marker proteins LC3-II and p53 were increased significantly in cells infected with pAd-HA-Numb compared with pAd-HA infected cells after Cisplatin administration (Figures 3(b) and 3(c)). Overexpression of Numb enhanced Cisplatin-induced autophagy, which was confirmed by immunofluorescence detection for LC3 and LAMP1 (Figures 3(d) and 3(e)). These data further confirmed that Numb may activate autophagy through p53 pathway.Figure 3
Overexpression Numb promotes autophagy in NRK-52E cells induced by Cisplatin. NRK-52E cells were infected with Ad-ctrl or Ad-Numb followed by treated with Cisplatin or vehicle (saline) for 6 hours. (a) The overexpression of Numb was confirmed using Western blotting assay.∗P<0.05 versus cells treated with vehicle. (b) The protein expression of Numb, p53, and LC3-II in cells of different groups as indicated, GAPDH was used as loading control. (c) Relative expression of Numb, p53, and LC3-II to GAPDH was represented in graphic. (d) Representative immunofluorescence images of co-expression of LC3-II (green) and LAMP1 (red) in cells of different groups as indicated. Magnified 400 times. (e) Quantitative statistics for immunofluorescence images. Data are shown as means±SD of three independent experiments. ∗P<0.05, ∗∗P<0.01 versus cells treated with saline-treated cells (Control). ∗P<0.05 versus vehicle-treated cells. #P<0.05, ##P<0.01 versus Ad-Ctrl infected cells with Cisplatin treatment. Data are shown as means±SD. n =3. ∗P<0.05 versus cells treated with saline (Control).
(a)(b)(c)(d)(e)


## 3.4. Numb Activates Cisplatin-Induced Autophagy in p53-Dependent Pathway

In terms of mechanism, to explore the role of p53 in autophagy promoted by Numb, NRK-52E cells infected with Ad-Numb were treated with an inhibitor of p53 as pifithrin-α (PIF-α) for 24 h. Western blotting showed that the protein expression of p53 was decreased significantly in PIF-α-treated cells compared with those treated with vector. Correspondingly, the autophagy-related LC3-II protein expression was decreased in PIF-α-treated cells significantly compared with vector-treated cells (Figure 4). These data indicated Numb-activated autophagy induced by Cisplatin in proximal tubular cells via p53-dependent pathway.Figure 4
Numb activates Cisplatin-induced autophagy in p53-dependent pathway. NRK-52E cells were infected with Ad-Numb for 24 h, then incubated with 20μM PIF-α or vehicle (DMSO) for 1 hour and followed by Cisplatin incubation for 6 hours. (a) The protein expression of Numb, p53, and LC3-II in cells of different groups as indicated, GAPDH was used as loading control. (b) Relative expression of Numb, p53 ,and LC3-II to GAPDH was represented in graphic. Data are shown as means±SD. n =3. ∗P<0.05 DMSO-treated Ad-Numb-infected cells with Cisplatin incubation.
(a)(b)


## 4. Discussion

It has been reported in a large number of studies that autophagy is involved in many human diseases, especially cancers. The role of autophagy in different types of cancers may vary and there are multiple autophagy markers such as LC3, Beclin1, and ATG7. Inhibition of autophagy with decreased LC3 expression tends to suppress epithelial-mesenchymal transition of lung cancer cells [35]. Beclin-1-mediated autophagy is reported positively related to better prognosis in uveal melanoma [36], while ATG7 involved in autophagy correlated with increased survival in malignant pleural mesothelioma [37]. Autophagy accompanied with immune escape is also related to many human diseases including cancers, such as glioma [38] and lung adenocarcinoma [39]. Moreover, the side effects of chemotherapy drugs for cancers always induced organ damage including AKI.AKI induced by Cisplatin is generally characterized as tubular death including apoptosis and necrosis [7, 8]. Autophagy is rapidly upregulated in proximal tubules in AKI induced by Cisplatin and plays a critical role in regulating proximal tubular death in AKI [19]. Our previous studies demonstrated that Numb attenuated proximal tubular death in AKI induced by Cisplatin [7, 8]. However, the effect and mechanism of Numb in autophagy of proximal tubular in AKI have not been clear so far. In our study, a well-established AKI model induced by Cisplatin and experiments in vitro was used to explore the effect and mechanism of Numb in tubular autophagy. Firstly, we found that the protein levels of Numb along with autophagy-related LC3-II and Beclin-1 were upregulated at the same time in AKI model mice induced by Cisplatin. Knockdown expression of Numb specifically in proximal tubular cells inhibited the activation of Cisplatin-induced autophagy both in vitro and in vivo, which suggested that Numb is a promotor of autophagy in AKI. Our results indicated that Numb promoted the activation of tubular autophagy in a p53-dependent pathway but not in a mTORC1-dependent pathway. Our study provides a novel role and mechanism action of Numb in autophagy in Cisplatin-induced AKI. As acts as a “double-edged sword,” autophagy could aggravate or ameliorate kidney injury. It has been showed in previous studies that autophagy can be activated in AKI and acts as a critical defense mechanism to alleviate AKI [30, 34]. Meanwhile, results of other studies indicated that autophagy may promote cell death in AKI [40]. The function of autophagy in tubular injury in AKI induced by Cisplatin remains contradictory. Unequivocal understanding of the role and molecular mechanisms of tubular autophagy is pivotal for unfolding the pathogenesis of AKI, which may provide therapeutic targets for AKI treatment. Numb has been reported positively associated with autophagic flux through regulating the function of lysosomes [25]. In line with this, the results of our study indicated that autophagy was activated in AKI model mouse induced by Cisplatin, which was significantly suppressed by Numb-siRNA both in vitro and in vivo, manifesting Numb-activated proximal tubular autophagy in Cisplatin-induced AKI model. Therefore, our results further confirmed that moderate activation of autophagy in tubules protected tubular from injury in AKI, while excessive activation of autophagy in tubules severed tubular injury in AKI.How Numb regulates proximal tubular autophagy in AKI model induced by Cisplatin was explored in our further study. Previous studies indicate that the pathway of mTOR-mediated autophagy is one of the best studied mammalian mechanisms for autophagy [14, 41]. We then explored whether Numb-activated tubular autophagy was mediated by inhibiting mTOR pathway in AKI induced by Cisplatin in preliminary experiments. Our results indicated that the activity of mTORC1 was significantly inhibited in PT-Nb-KO mice (Numb knockout in proximal tubules) kidneys as well as in Numb-siRNA-transfected NRK-52E cells, indicating that Numb-activated autophagy was not mediated by mTORC1. Consistently, Susan E. Quaggin et al. demonstrated that Podocyte-specific deletion of mTOR suppressed autophagic flux and resulted in heavy proteinuria in 3 weeks old mice [42]. In addition, Xiaofeng Jia et al. reported that Trehalose attenuates spinal cord injury through activating autophagy mTOR independently [43]. Together with our current findings, all the results highlight an important role of mTORC1-independent pathway in cell autophagy during renal injury or other diseases.p53 also takes a critical part in regulating tubular autophagy [17–19]. Zheng Dong et al. reported that p53 may regulate autophagy positively in Cisplatin-treated renal tubular cells [19]. Xiaodong et al. demonstrated that p53 can induce autophagy by upregulating ULK1 in camptothecin-treated U2OS cells [44]. In our study, it was found that Numb promoted the activation of autophagy in Cisplatin-treated NRK-52E cells as well as the upregulation of p53. Moreover, pharmacologically suppressing the p53 activity inhibited autophagy in Numb-overexpressed cells. Therefore, our results suggested Numb-activated proximal tubular autophagy through activating p53 pathway. Collectively, consistent with the study of Zheng Dong, our study further confirmed that p53-mediated autophagy protects the kidney from injury induced by Cisplatin.The mechanism of AKI is considered complicated which is concerned with dysregulation of autophagy and immune response [45]. It has been reported in a previous study that Numb participated in regulating immune response in chronic Q fever [46]. In this study, we only focused on clarifying the role of Numb in regulating autophagy but not immune response in AKI, and only one cell line was involved in the experiments, which would be involved in our further study.In conclusion, our data demonstrated that Numb promoted proximal tubular autophagy activation mediated by p53 in the AKI model induced by Cisplatinin vitro and in vivo, which unfold a novel molecule in the effect and regulatory mechanism of tubular autophagy in AKI and may provide a target for the treatment of AKI.



---

*Source: 8213683-2022-06-27.xml*