Lipophagy deficiency exacerbates ectopic lipid accumulation and tubular cells injury in diabetic nephropathy

Autophagy-mediated lipotoxicity plays a critical role in the progression of diabetic nephropathy (DN), but the precise mechanism is not fully understood. Whether lipophagy, a selective type of autophagy participates in renal ectopic lipid deposition (ELD) and lipotoxicity in the kidney of DN is unknown. Here, decreased lipophagy, increased ELD and lipotoxcity were observed in tubular cells of patients with DN, which were accompanied with reduced expression of AdipoR1 and p-AMPK. Similar results were found in db/db mice, these changes were reversed by AdipoRon, an adiponectin receptor activator that promotes autophagy. Additionally, a significantly decreased level of lipophagy was observed in HK-2 cells, a human proximal tubular cell line treated with high glucose, which was consistent with increased lipid deposition, apoptosis and fibrosis, while were partially alleviated by AdipoRon. However, these effects were abolished by pretreatment with ULK1 inhibitor SBI-0206965, autophagy inhibitor chloroquine and enhanced by AMPK activator AICAR. These data suggested by the first time that autophagy-mediated lipophagy deficiency plays a critical role in the ELD and lipid-related renal injury of DN.


INTRODUCTION
Diabetic nephropathy (DN) is a severe complication of diabetes mellitus and has become the most common primary disease leading to end-stage renal disease worldwide [1]. The importance of tubular injury in the progression of DN and the role of inflammation, mitochondrial oxidative stress and autophagy disorders in the pathogenesis of DN was confirmed in our previous studies [2,3]. Emerging evidence suggests that the dyslipidemia in patients with DN was associated with estimated glomerular filtration rate (GFR), inflammation, fibrosis and disease progression [4,5]. High-plasma triacylglycerols(TG) and nonesterified fatty acids (NEFAs) results in the lipid accumulation and lipotoxicity in non-adipose tissues [6,7]. Moreover, renal cells were damaged by ectopic lipid deposition (ELD) associated with lipotoxicity via activating inflammation, reactive oxygen species (ROS) production, mitochondrial dysfunction and cell-death in kidney of DN [4,[8][9][10][11][12]. However, the precise mechanism through which lipid disorders impact the kidney damage in DN remains unclear.
Selective autophagy, lipophagy, plays a key role in regulating the synthesis and degradation of intracellular lipid droplets (LDs) in hepatocytes [13], macrophage foam cells [14], etc. Lipophagy refers to that when LDs are sequestered by autophagosomes, which fuse with lysosomes to form autolysosomes, TG and cholesteryl esters in LDs are then degraded by lysosomal hydrolases in autolysosomes into free fatty acids (FFAs) that are recycled back into the cytosol for mitochondrial β-oxidation [15,16]. The activation of lipophagy was dependent on the environment and energy requirement of different types of cells [17], such as during prolonged starvation and the early phase of lipid challenge [16,18]. Defective lipophagy were reported to be linked with liver steatosis, obesity, atherosclerosis and the metabolic syndrome of aging [14,[18][19][20][21]. Interestingly, increased lipophagy may relieve insulin resistance by decreasing lipid accumulation in adipocytes in patients with type 2 diabetes [22]. However, the level of lipophagy in kidney tissues in DN and whether it is involved in the kidney injury is unknown.
In this study, lipophagy deficiency was observed in the tubular cells of patients with DN and db/db mice, which was accompanied by significantly ELD, oxidative stress, apoptosis. These damages were ameliorated in tubular cells as the levels of lipophagy was increased by AdipoRon administration, while the effects were blocked partially by AdipoR1 siRNA, autophagy inhibitor, ULK1 inhibitor and enhanced by AMPK activator. These data demonstrated by first time that disrupted lipophagy modulated by AdipoR1/AMPK pathway plays a key role in ELD and lipotoxicity in tubular cells of DN.
24-h urine protein and decreased albumin were observed in patients with DN (Table 1). Significant typical nodular glomerulosclerosis, basement membrane thickening, focal tubular atrophy, interstitial fibrosis, severe tubular injury and obvious lipid deposition occurred in the kidney of patients with DN ( Fig. 1A-D). Positive correlations were observed in tubular interstitial damage and lipid deposition (r = 0.872), tubular interstitial damage and adipose differentiation related protein (ADRP) expression (r = 0.907), lipid deposition and ADRP expression (r = 0.807) in kidney tissues of patients. Transmission Electron Microscope (TEM) detection showed that some LDs were sequestrated by doublemembrane organelles, namely, autophagosomes, in both groups. More LDs and less lipophagy were represented in the kidney of DN, which indicated that lipid deposition and insufficient lipophagy may be closely related to pathological injury in the kidney of DN (Fig. 1E).
The expression of AdipoR1, p-AMPK, and LC3B in the kidneys of patients with DN were noticeably decreased and the AdipoR1 expression was correlated with renal function as well as autophagosome assembly Moreover, considering that AdipoR1 is involved in the regulation of lipid metabolism and autophagy, AdipoR1 was detected to explore the mechanism of lipophagy deficiency in the DN kidney tissues. Immunofluorescence (IF) analysis revealed that AdiopR1 expression was reduced in kidneys of patients with DN (Fig. 1F, G). Additionally, AMPK is an important modulator of autophagy and a downstream regulator of AdipoR1, the level of its phosphorylation in the kidney and its relationship with lipid deposition and tubular injury were detected. The expression of p-AMPK and LC3B in the kidneys of patients with DN was noticeably decreased (Fig.1F, G). Here, AdipoR1 expression was negatively correlated with lipid deposition (r = −0.653), ADRP expression (r = −0.683), and positively correlated with p-AMPK expression (r = 0.849) and LC3B expression (r = 0.804). P-AMPK expression was negatively correlated with tubular interstitial damage (r = −0.868), lipid deposition (r = −0.774), ADRP expression (r = −0.863), but positively correlated with LC3B expression (r = 0.893). LC3B expression was negatively correlated with tubular interstitial damage (r = −0.761) and lipid deposition (r = −0.618). Meanwhile, Gene Ontology analysis was carried out, and the results showed that some of the genes positively coexpressed with AdipoR1 (correlation coefficient ≥0.60) were enriched in metabolic pathway and autophagosome assembly according to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway ( Fig. 2A) and biology process analysis (Fig. 2B), respectively. Additionally, real-time PCR detection determined the expression of AdipoR1, AdipoR2 and AdipoQ in the HK-2 cells, BUMPT cells (a mouse proximal tubulederived cell line) and mouse podocytes, AdipoR1 and AdipoR2 were expressed in all of these cells' line, while AdipoQ was expressed in BUMPT cells and mouse podocytes (Fig. 2C). Moreover, according to the Nephroseq database, AdipiR1 expression was positively correlated with GFR (r = 0.708, Fig. 2D) and negatively correlated with serum creatinine (Scr) level (r = −0.644, Fig. 2E) in patients with DN, indicating that the expression of AdipoR1 was closely correlated with renal function in patients with DN.
Increased lipophagy by AdipoRon ameliorates renal injury, lipid deposition, oxidative stress, and apoptosis in the kidney of db/db mice Treatment of db/db mice with AdipoRon for 4 weeks, the body weight, kidney weight and Scr level of db/db mice showed no significant change from those of the untreated group. Blood glucose, HbA1C, TG and 24-h albuminuria were increased in db/db mice compared with control mice, but these changes were all reversed by AdipoRon treatment. Serum adiponectin levels decreased obviously in db/db mice compared with control mice, and there was no significant difference in adiponectin levels between the db/db mice treated with AdipoRon and the untreated db/db mice ( Table 2).
In addition, glomerular hypertrophy, mesangial cells and matrix significantly increased in some glomeruli and some dilated or atrophied tubules and renal interstitial fibrosis were noted in the kidneys of db/db mice, which were all partially alleviated following AdipoRon treatment (Fig. 3A, C). Additionally, cell apoptosis and oxidative stress in the kidneys were enhanced in db/db mice compared with control mice, while were attenuated by AdipoRon (Fig. 3B, D). Moreover, AdipoRon administration decreased intrarenal lipid concentrations, which were represented by the decreased renal NEFA and TG in db/db mice (Fig. 3E, F), while there was no significant alteration in TC (Fig. 3G). Further analysis revealed lipid deposition in the kidneys of db/db mice via IHC for ADRP detection and Oil-Red O staining. AdipoRon dramatically ameliorated this enhancement (Fig. 3H, I). Ultrastructural analysis revealed an increased number of LDs in the kidneys of db/db mice, and fewer LDs in the kidneys of db/db mice treated with AdipoRon. Importantly, in the kidneys of control mice and db/db mice treated with AdipoRon, clear autophagosomes or autolysosomes that engulfed LD were noted (Fig. 3J, K).
AdipoRon upregulated the expression of p-AMPK, p-ULK1 and autophagosome-associated proteins, while reduced fibrosis in the kidney of db/db mice The decreased expression of AdipoR1, p-AMPK and LC3B and increased p-mTOR level in db/db mice were all partially reversed by AdipoRon treatment (Fig. 4A-D). Similar alterations were also showed in autophagosome-associated proteins such as beclin1 (BECN1), autophagy related 5 (ATG5) and LC3-II in the LD fraction. Sequestosome 1 (SQSTM1) was not detected. The purification degree of the LD fraction was  determined by the abundant ADRP expression and the absence of GAPDH (Fig. 4E). Fibronectin (FN)and Collagen I were reduced by AdipoRon treatment (Fig. 4F, G). Protein alterations in ADRP, sterol regulatory element binding protein-1 (SREBP-1), FN and Collagen I were further confirmed by WB analysis (Fig. 4H, I).
AdipoRon reversed the lipophagy alteration and lipid deposition in HK-2 cells exposed to high-glucose (HG) ambience The co-localization studies of autophagosomes, lysosomes and LDs were performed to identify whether LDs are associated with lipophagy. AdipoRon treatment increased autophagosomes (stained by monodansylca-daverine, MDC) and lysosomes (stained by Lyso-tracker), while decreased the number of LDs (stained by Bodipy). Moreover, AdipoRon treated cells exhibited more co-localization of LDs with autophagosomes and lysosomes (increased white stained regions). Additionally, inhibition of lysosomal degradation of LDs that sequestered autophagosomes by chloroquine (CQ) enhanced co-localization of LDs with autophagosomes (Fig. 5A). The upregulation of LC3-II levels in isolated LD fraction from HK-2 cells exposed to HG conditions with AdipoRon and CQ treatment were confirmed by WB analysis (Fig. 5B, E). Similar results were also seen in the expression of transcription factor EB(TFEB) in nuclear protein extracted from HK-2 cells under HG condition pretreated with AdipoRon (Fig. 5C, D). Additionally, a decreased expression of AdipoR1, p-AMPK and p-ULK1 as well as an increasing level of p-mTOR in total cell protein from HK-2 cells exposed to HG were found, while were reversed when pretreated with AdipoRon (Fig. 6A, B).
The effects of AdipoRon on increasing lipophagy, reducing ELD and fibrosis in HK-2 cells were partially blocked by AdipoR1 siRNA and ULK1 inhibitor, while enhanced by AMPK activator Effects of AdipoRon were abolished by pretreatment with AdipoR1 siRNA or ULK1 inhibitor SBI-0206965 and reinforced by AMPK activator AICAR (Fig. 6C, D). Similar alterations in BECN1, ATG5 and LC3-II were also observed in the LD fraction isolated from HK-2 cells exposed to HG after pretreated with AdipoR1 siRNA, SBI-0206965 and AICAR (Fig. 6E). When intracellular LDs were engulfed by autophagosomes, ADRP was removed and dissociated from RAB7, the protein on the LD surface that subsequently interacts with autophagy proteins, such as SQSTM1/p62 and LC3-II [16]. The co-localization of RAB7 and LC3B, BECN1 and LDs (Bodipy stained) was decreased in HK-2 cells under HG conditions, and AdipoRon administration reversed these changes. However, these effects were partially abolished by pretreatment with AdipoR1 siRNA, SBI-0206965 and enhanced by AICAR (Fig. 7A, B). Similar alterations were also found in the intracellular lipid deposition (Fig. 7C, D), which was accompanied by the protein alterations of ADRP, SREBP-1, FN and Collagen I (Fig. 7E, F). Overall, AdipoRon activated AdipoR1/AMPK pathway to increase lipophagy that alleviated lipid deposition and fibrosis in HK-2 cells exposed to HG conditions pathway.

DISCUSSION
The present study demonstrated by first that the lipophagy deficiency in tubular cells was related to the ELD-associated kidney damage in both patients and mice with DN. Increasing the level of lipophagy by administration of AdipoRon through AdipoR1/AMPK pathway, which ameliorates lipotoxicityassociated tubular injury, oxidative stress and apoptosis. These data indicate that lipophagy dysfunction contributes to ELDinduced kidney injury in DN.
Lipophagy, a type of selective autophagy that sequesters LDs in autophagosomes for degradation by lysosomes [15]. It has been reported that lipophagy was associated with hepatic disorders, including liver steatosis, nonalcoholic fatty liver disease, and liver fibrosis [23,24]. The methods of identifying lipophagy include Oil-Red O, BODIPY 493/503 or HCS LipidTOX staining, triglyceride content determination and TEM [15]. It also has been demonstrated that lipophagy is induced to breakdown LDs to maintain energy homeostasis under prolonged starvation in proximal tubule cells [15,25]. Furthermore, lipophagy was inhibited under nutrient abundance since FFAs were not needed as an energy source for cells [26]. Chronic lipid exposure such as ELD might be a contributor to lipophagy disorder in type 2 diabetes [22]. Moreover, previous studies by our and other group have found that renal ELD and its associated lipotoxicity in the kidney are the main causes of the development of obesity-related nephropathy and type 2 DN [4,[27][28][29]. Abnormal autophagy was found to participate in kidney injury in DN in vivo and in vitro [25,30,31]. However, whether autophagy-mediated lipophagy involved in the pathogenesis and development of chronic kidney disease including DN still needs to be discussed. Here, we demonstrated that the reduced lipophagy and lipid deposition in the renal tissues of patients with DN were positively correlated with lipotoxicity induced tubular injury (Fig. 1).
On the other hand, decreased renal AdipoR1 expression was accompanied by increased renal TG levels in diabetic rats [32], while increasing AdipoR1 expression ameliorated lipotoxicity [33]. Here, we confirmed the expression of AdipoR1 in tubular cells, and Gene Ontology analysis showed that the genes positively correlated with AdipoR1 expression were enriched in metabolic pathways and autophagy formation, and the expression level of AdipoR1 was correlated with renal function in DN patients (Fig. 2). In addition, AdipoRon, an AdipoR1 activator, which can enhance glucose and lipid metabolism and inhibits lipotoxicity of diabetic mice through AdipoR1/ AMPK pathway [10,34], it also might promote autophagy in renal epithelial cells [35]. However, it is not clear that whether enhanced autophagy-mediated lipophagy by AdipoRon might alleviate lipotoxicity induced renal injury in DN. In this study, we found that levels of lipophagy in kidney of diabetic mouse was increased by AdipoRon, which was accompanied by decrease in proteinuria and renal lipid contents levels as well as    AdipoRon increased lipophagy in HK-2 cells exposed to HG environment. Representative images of intracellular autophagosome (MCD), lysosomes (Lyso-tracker) and lipid (Bodipy) co-localization in HK-2 cells in an HG environment with or without AdipoRon, CQ pretreatment (A). WB analysis of LC3II expression using HOM and isolated LD fractions from HK-2 cells exposed to HG conditions with or without different AdipoRon concentrations (B). WB and densitometric analysis of TFEB expression using nuclear protein from HK-2 cells exposed to HG conditions with or without AdipoRon (C, D). WB analysis of LC3II expression using isolated LD fractions from HK-2 cells exposed to HG conditions with or without AdipoRon and CQ pretreatment (E). Values are the mean ± SD; *p < 0.05 versus LG group, # p < 0.05 versus HG group. n = 3.
the alleviation of renal pathological injury, oxidative stress, apoptosis and fibrosis (Table 2 and Figs. 3 and 4). These data further demonstrated that pharmacological enhancement of the level of autophagy-mediated lipophagy could reverse the ELD and tubular cells injury in DN. It is reported that AdipoR1 activation by AdipoRon could restore AMPK-mediated autophagosome formation and stimulate autophagosome clearance under diabetic conditions [36]. Additionally, the decreased AdipoR1 and AMPK activation led to a reduction in fatty acid oxidation and an increase in fatty acid synthesis, promoting the progression of type 2 diabetes [37,38]. The increased AMPK phosphorylation and subsequently activated autophagy has been demonstrated to be a renal protection mechanism in DN [39,40]. AMPK is a key enzyme that regulates autophagy, which is involved in the development of DN [41]. Furthermore, it is known that autophagy was promoted by AMPK through directly activating ULK1 phosphorylation, while inhibited by mTOR through disrupting the interaction between ULK1 and AMPK [42]. Additional, ULK1 plays an important role in regulating lipid metabolism in adipocytes by regulating autophagy [43]. Increased levels of p-AMPK and ULK1 were suggested to be related to the activation of lipophagy in hypothalamic neurons [44]. As expected, treatment of HK-2 cells with AdipoRon reversed HG-induced lipophagy dysfunction and increased lipid deposition, while these effects were partially blocked by pretreatment with autophagy inhibitor, but enhanced by AMPK activtor (Figs. 5-7).  6 AdipoRon increased lipophagy through AdipoR1/AMPK pathway. WB and densitometric analysis of AdipoR1, p-mTOR, p-AMPK and p-ULK1 expression in HK-2 cells exposed to HG conditions with or without different AdipoRon concentrations (A, B). WB and densitometric analysis of AdipoR1, p-mTOR, p-AMPK and p-ULK1 expression in HK-2 cells exposed to an HG environment pretreated with AdipoRon, AdipoR1 siRNA, AICAR or SBI-0206965 (C, D). WB analysis of BECN1, ATG5, LC3 and SQSTM1 using isolated LD fractions from HK-2 cells exposed to HG conditions with or without AdipoRon, AdipoR1 siRNA, AICAR and SBI-0206965 (E). Values are the mean ± SD; *p < 0.05 versus LG group, # p < 0.05 versus HG group, & p < 0.05 versus HG + AdipoRon group. n = 3. Fig. 7 The effects of AdipoRon on increasing lipohagy and alleviating lipid deposition and fibrosis were partially blocked by ULK1 inhibitor, while enhance by AMPK activator. Confocal IF images showing the co-localization of RAB7 and LC3B and the co-localization of BECN1 and LDs (Bodipy) in HK-2 cells exposed to HG conditions and pretreated with or without AdipoRon, AdipoR1 siRNA, AICAR and SBI-0206965 (A, B). Representative images and semiquantification of the lipid deposition in HK-2 cells exposed to an HG environment pretreated with AdipoRon, AdipoR1 siRNA, AICAR and SBI-0206965 (C, D). WB and densitometric analysis of ADRP, SREBP-1, FN and Collagen I expressions in HK-2 cells exposed to HG conditions with or without AdipoRon, AdipoR1 siRNA, AICAR and SBI-0206965 (E, F). Values are the mean ± SD; *p < 0.05 versus LG group, # p < 0.05 versus HG group. n = 3.
These data suggested that increased lipophagy by AdipoRon in tubular cells of DN might through activating AdipoR1/AMPK pathway.
Briefly, in this study we found that the lipophagy deficiency plays a critical role in the ELD and lipotoxicity in tubular cells of DN. AdipoRon can reduce intrarenal lipotoxicity-associated renal injury and fibrosis in DN through increasing lipophagy by activating AdipoR1/AMPK pathway. These findings suggest that targeting lipophagy might be a potential therapeutic strategy for DN and other ELD-related metabolic diseases.

Clinical data
Patients with DN were newly diagnosed by renal biopsy who used insulin to control blood glucose. Patients using lipid-lowering drugs, RAS inhibitors before biopsy and those with urinary tract infections, or with inflammatory, neoplastic, cardiovascular, hepatic, renal, lung or neuroendocrine diseases were excluded. The patients in the control group were the first diagnosed patients with primary glomerulonephritis of varying degrees of proteinuria and the pathological diagnosis was minimal change nephropathy. These patients were not treated prior to pathological diagnosis. The DN group included 15 patients (8 males and 7 females), the average age is 43.69 ± 7.62 years (34-57 years) and the control group included 15 patients (8 males and 7 females), the average age is 41.54 ± 7.66 years (32-56 years). The clinical experimental procedures were approved by the Ethics Committee of Second Xiangya Hospital, Central South University, and informed consent was obtained from all the patients.

Animal experimental design
Fourteen-week-old male C57BLKS/J db/db and C57BLKS/J db/m mice were purchased from the Aier Matt Experimental Animal Company (China). They were randomly divided into the following three groups: a db/m group (control, n = 12), a db/db group receiving intragastric injection with AdipoRon (n = 12) and a db/db group that receive intragastric injection with 0.5% sodium carboxymethyl cellulose solution, which served as vehicle control (n = 12). AdipoRon (30 mg/ kg) was dissolved in 0.5% sodium carboxymethyl cellulose solution and then provided to db/db mice once daily via oral gavage from 16 weeks of age for 4 weeks as described previously [45]. The mice were euthanized at 20 weeks of age. All animal procedures were approved by The Animal Care and Use Committee of Second Xiangya Hospital of Central South University.

Assessment of physiological features
Body weights and blood glucose levels were measured twice per week. Twenty-four-hour urine samples were collected using metabolic cages before the mice were euthanized. Urine albumin concentrations were tested using an Albuwell M kit, and levels of serum creatinine were measured using HPLC as previous study [45]. The intrarenal lipid levels, namely FFAs, TG and TC using commercial kits (Wako, Osaka, Japan) according to manufacturer's instructions.

IHC of renal tissue
Four-micrometer -thick paraffin-embedded renal tissue sections from human or mice were prepared for IHC studies, as previously described [3]. The sections were deparaffined in xylene, rehydrated in ethanol and soaked in 10 mM citrate buffer at 65°C for antigen retrieval. After blocking endogenous peroxidase activity with 3% H 2 O 2 , the sections were exposed to 5% bovine serum albumin (BSA) and sequentially incubated with antibodies against ADRP (1:100, Proteintech, 15294-1-AP) overnight at 4°C. The sections were then incubated with secondary antibodies conjugated with peroxidase, treated with diaminobenzidine, and finally counterstained with hematoxylin. Images were obtained using a Nikon microscope and analyzed by ImageJ.

Lipophagy analysis
Lipophagy was monitored with the following methods. (1) Oil-Red O staining: Five-micrometer-thick, 4% PFA-fixed, frozen renal tissue sections or 4% PFA-fixed HK-2 cells were rinsed with 60% isopropanol for 20-30 seconds, and stained with Oil-Red O dye solution for 15 min as previously described [12]. (2) TEM: Kidney samples were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4°C and fixed with 1% osmium tetroxide solution for 1 h. The samples were then dehydrated in ethanol and embedded in Epon 812. Ultrathin sections (~80 nm) were prepared and stained with uranyl acetate and lead citrate, followed by observation of the LD-associated double-membrane structure using TEM. (3) WB analysis: Lipid droplet components were isolated from mouse kidney samples and HK-2 cells as previously described [48]. Briefly, kidney samples from 8 kidneys were homogenized by ultrasonic disruption after being kept in icy buffer A (20 mM tricine, 250 mM sucrose, PH = 7.8) plus 0.2 mM phenyl methane sulfonyl fluoride (PMSF) for 20 min. Then, the samples were centrifuged at 100 × g for 10 min at 4°C, and the supernatant was disrupted under high pressure. The homogenate was centrifuged at 3000 × g for 10 min at 4°C to obtain the postnuclear supernatant (PNS) fraction. Ten milliliters of PNS and two milliliters of buffer B (20 mM HEPES, 100 mM KCl and 2 mM MgCl2, pH = 7.4) were placed into a SW40 tube and centrifuged by an ultracentrifuge at 100,000 × g for 60 min at 4°C. The crude LD fraction was carefully collected from the top to the Eppendorf tube. Finally, the crude LD fraction was resuspended in 200 μL of buffer B in the Eppendorf tube and centrifuged at 20,000 × g for 5 min at 4°C twice to purify the LD fraction. For HK-2 cells, cells were washed with ice-cold PBS before collection, followed by centrifugation at 1000 × g for 10 min at 4°C. The samples were then suspended in buffer A plus 0.2 mM PMSF and incubated on ice for 20 min, followed by ultrasonic disruption. After centrifugation at 3000 × g for 10 min at 4°C, and the PNS fraction and buffer B were transferred into a SW40 tube to centrifuge at 182,000 × g for 60 min at 4°C. The LD fraction was carefully collected from the top band of the gradient and sequentially washed and purified three times by buffer B as mentioned above. Finally, an equal amount of the LD protein was analyzed by WB analysis. (4) Confocal microscopy: Confocal microscopy was carried out to detect the colocalization of LDs (BODIPY 493/503 neutral lipid stains), autophagosomes (MDC stains) and lysosomes (Lyso-tracker stains) the co-localization of RAB7 and LC3B and the co-localization of BECN1 and LDs (BODIPY 493/503 neutral lipid stains) in HK-2 cells. The analysis was performed using LSM 510 software (Zeiss).

Extraction of nuclear protein
Separation of nuclear protein from the cytoplasmic protein was carried out using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer's instructions. Briefly, HK-2 cells were resuspended in ice-cold hypotonic lysis buffer after intervention, and subcellular protein fractions extracted by homogenizing and centrifuging. Finally, an equal amount of the nuclear protein was analyzed by WB analysis. Primary antibodies specific for TFEB (1:1000, Proteintech, 13372-1-AP) were used. The bands were evaluated using a Tanon 5200 Multi instrument (Tanon Instruments, China). The band densities of target proteins were compared with those of Lamin B (1:1000, Proteintech, 66095-1-Ig) by using ImageJ.