Abstract

Objective

   This study investigates the effects of caloric restriction (CR) on
   renal injury and fibrosis following ischemia-reperfusion injury (IRI),
   with a focus on the roles of the mechanistic/mammalian target of
   rapamycin complex 1 (mTORC1) signaling and autophagy.

Methods

   A mouse model of unilateral IRI with or without CR was used. Renal
   function was assessed through serum creatinine and blood urea nitrogen
   levels, while histological analysis and molecular assays evaluated
   tubular injury, fibrosis, mTORC1 signaling, and autophagy activation.
   Inducible renal tubule-specific Atg7 knockout mice and autophagy
   inhibitor 3-MA were used to elucidate autophagy's role in renal
   outcomes.

Results

   CR exacerbated renal dysfunction, tubular injury, and fibrosis in IRI
   mice, associated with suppressed mTORC1 signaling and enhanced
   autophagy. Rapamycin, an mTORC1 inhibitor, mimicked the effects of CR,
   further supporting the involvement of mTORC1-autophagy pathway.
   Tubule-specific Atg7 knockout and autophagy inhibitor 3-MA mitigated
   these effects, indicating a central role for autophagy in CR-induced
   renal damage. Glucose supplementation, but not branched-chain amino
   acids (BCAAs), alleviated CR-induced renal fibrosis and dysfunction by
   restoring mTORC1 activation. Finally, we identified leucyl-tRNA
   synthetase 1 (LARS1) as a key mediator of nutrient sensing and mTORC1
   activation, demonstrating its glucose dependency under CR conditions.

Conclusion

   Our study provides novel insights into the interplay between nutrient
   metabolism, mTORC1 signaling, and autophagy in IRI-induced renal
   damages, offering potential therapeutic targets for mitigating
   CR-associated complications after renal IRI.

   Keywords: Acute kidney injury, Caloric restriction, mTORC1,
   Ischemia-reperfusion injury, Autophagy

1. Introduction

   Acute kidney injury (AKI) is a significant clinical problem with high
   morbidity and mortality, often resulting in permanent kidney damage and
   progression to chronic kidney disease (CKD) [[39]1].
   Ischemia-reperfusion injury (IRI) is a common cause of AKI, leading to
   oxidative stress, inflammation, and tubular damage [[40][2], [41][3],
   [42][4]]. Even after severe injury, the kidneys have the potential to
   recover. Renal tubular epithelial cells have the ability to
   dedifferentiate, proliferate, and redifferentiate to repair damaged
   nephrons. However, maladaptive tubular repair can result in renal
   dysfunction and structural damage, leading to CKD, a process influenced
   by the severity of the injury and the capacity for recovery.

   Caloric restriction (CR) is a well-established intervention known to
   extend lifespan and confer protection against various metabolic
   diseases in multiple organisms [[43][5], [44][6], [45][7]]. CR exerts
   its effects by modulating key metabolic pathways, including the
   inhibition of the mechanistic target of rapamycin (mTOR) and the
   activation of autophagy, a cellular process that degrades damaged
   organelles and proteins [[46]8,[47]9]. While CR has been shown to be
   beneficial in metabolic health and weight management, recent studies
   have revealed its adverse effects on wound healing, infection
   [[48]10],and certain types of tissue regeneration [[49]11]. Currently,
   the impact of caloric restriction on tissue regeneration following AKI
   remains unclear.

   In response to nutrient availability, the mTORC1 pathway plays a
   central role in regulating cellular growth, metabolism, and survival
   [[50]12,[51]13]. The activation of mTOR in renal tubular cells after
   AKI is essential for tubule repair [[52]14,[53]15], although several
   reports suggest that mTOR inhibition can also have protective effects
   [[54]16]. Therefore, the role of mTOR in AKI is complex and may be
   context-dependent. Additionally, mTOR influences autophagy, a process
   generally considered crucial in the context of chronic kidney disease
   [[55]17,[56]18]. Although finely regulated autophagy is essential for
   kidney repair and regeneration, recent studies suggest that excessive
   or dysregulated autophagy could exacerbate tissue injury under fibrotic
   conditions [[57]19,[58]20].

   In this study, we aimed to explore the effects of CR on renal injury
   and fibrosis following IRI and to uncover the underlying mechanisms.
   Surprisingly, we found that CR not only failed to mitigate the
   progression of AKI to CKD but also exacerbated renal dysfunction and
   fibrosis during this process. Through omics analysis, we focused on the
   roles of mTORC1 signaling and autophagy to uncover the reasons behind
   this unexpected outcome. Our findings shed new light on the intricate
   interplay between metabolism, autophagy, and renal injury, offering
   valuable insights into the therapeutic implications of CR and mTORC1
   modulation in kidney disease.

2. Materials and methods

2.1. Animal models and treatments

   All animal experiments were approved by the Ethics Committee of Three
   Gorges University (202205010T2). Male C57BL/6 mice (8–10 weeks old)
   were purchased from the Experimental Animal Center of China Three
   Gorges University (Yichang, China). Autophagy reporter mice
   (CAG-RFP-GFP-LC3) were purchased from the Jackson Laboratory (stock No:
   027139). Following the experiment, CAG-RFP-GFP-LC3 mice were perfused
   with 4 % paraformaldehyde. The extracted kidneys were post-fixed
   overnight in the same fixative, equilibrated in 30 % sucrose, and
   embedded in Tissue-Tek® O.C.T. compound. Cryosections were then
   prepared for observation and image acquisition under a fluorescent
   confocal microscope. For the IRI model, mice were anesthetized with
   isoflurane, and the left kidney was subjected to ischemia by clamping
   the renal pedicle for 28 min, followed by reperfusion. The right kidney
   was removed via uninephrectomy at day 13 post-IRI, and mice were
   sacrificed on day 14 [[59]4,[60]21]. On the fourth day post-surgery,
   mice in the calorie restriction group were fed at 70 % of the normal
   intake, while control and sham-operated groups had ad libitum feeding.
   All groups had free access to water. Mice were monitored daily, and
   their condition was recorded. On day 13 of reperfusion, a right
   nephrectomy was performed, and 24 h later, blood was collected from the
   left renal artery.

   For autophagy inhibition studies, mice were treated with 3-MA
   (15 mg/kg, intraperitoneally) daily starting on day 4 post-IRI. For
   mTORC1 inhibition, mice received rapamycin (Rap) (2.5 mg/kg/day,
   intraperitoneally) starting on day 4 post-IRI. In the study on glucose,
   BCAA, aspartate, or asparagine supplementation, we administered
   0.14 g/ml of glucose or the respective amino acids via drinking water.

2.2. Gene delivery in vivo

   In the animal experiments, adenoviruses(Ad) carrying ribosomal protein
   S6 kinase, polypeptide 1(Rps6kb1) or its nonsense sequence (negative
   control, NC) (Sangon Biotech, Shanghai, China) were introduced into the
   mouse kidneys immediately following renal ischemia, as described
   previously [[61]22]. Briefly, 100 μL of adenovirus (10^8 transduction
   units/μL) was administered into the kidneys of anesthetized mice using
   a 30 G syringe. The needle was inserted at the lower pole of the kidney
   and advanced towards the upper pole, with the adenoviruses solution
   being infused slowly while the needle was gradually withdrawn.

2.3. Generation of inducible renal tubule-specific Atg7 knockout mice

   Male C57BL/6J mice were housed in the animal facilities at China Three
   Gorges University and were treated humanely according to guidelines of
   the Institutional Animal Use and Care Committee with free access to
   water and food. An inducible and conditional system was used to
   knockout Atg7 in the renal tubule of adult mice. Three transgenic mouse
   lines were cross-bred: Pax8-reverse tetracycline-dependent
   transactivator (Pax8-rtTA) mice(Jackson lab, stock No: 007176),
   tetO-Cre mice (Jackson lab, stock No: 006234) and Atg7 flox/flox
   mice(Jackson lab, stock No:034429). Tail DNA from all mice was
   genotyped by PCR analysis. Male mice aged 6–8 weeks were randomly
   assigned into different groups, with at least six mice per group: the
   non-postconditioning (Non CR) group with normal diet, calorie
   restriction group (CR). In the IRI model, mice were anesthetized with
   isoflurane, and ischemia was induced in the left kidney by clamping the
   renal pedicle for 28 min, followed by reperfusion. Starting on the
   fourth day post-unilateral IRI surgery, doxycycline (Sigma-Aldrich,
   D9891) was dissolved in drinking water containing 5 % sucrose and
   administered to the mice at a concentration of 2 mg/ml for one week to
   induce Atg7 deletion in renal tubules. Genotyping of genomic DNA
   extracted from kidney tissues was performed by PCR using primer Atg7s
   (5′- CTTCTGCAAGGCCCACTAAC-3′) and primer Atg7 96–121c (5′-
   CCCGGAGAAGTCTGAGTCTG-3′). The calorie restriction group was fed a
   limited diet, with daily food intake at 70 % of the normal level, while
   the control group was allowed ad libitum feeding after ischemia
   surgery, and the sham-operated group was also fed ad libitum. The
   condition of the mice was observed daily during restricted feeding, and
   records were kept. On day 13 of reperfusion, a right nephrectomy was
   performed, and 24 h later, the mice were anesthetized for blood
   collection from the left renal artery.

2.4. Renal function and histological analysis

   Serum creatinine (CRE) and blood urea nitrogen (BUN) levels were
   measured by the central laboratory of the Center People's Hospital of
   Yichang (Roche Diagnostics GmbH, Penzberg, and Mannheim, Germany).
   Kidney tissues were fixed in 10 % neutral-buffered formalin, embedded
   in paraffin, and sectioned at 4 μm for histological analysis. Sections
   were stained with hematoxylin and eosin (H&E) for general histology,
   Masson's trichrome for fibrosis, and Sirius Red for collagen
   deposition. Tubular injury was scored in a blinded fashion based on the
   percentage of affected tubules in randomly selected fields [[62]23].
   Interstitial fibrosis was quantified as the percentage of fibrotic area
   relative to the total area [[63]24].

2.5. Immunohistochemistry and immunofluorescence

   Paraffin-embedded kidney sections were deparaffinized, rehydrated, and
   subjected to antigen retrieval in citrate buffer (pH 6.0). Sections
   were blocked with 5 % bovine serum albumin and incubated overnight at
   4 °C with primary antibodies. Sections were incubated with
   HRP/AP-conjugated secondary antibodies and developed with DAB substrate
   or ImmPACT Vector Red Substrate Kit. For immunofluorescence, sections
   were incubated with fluorophore-conjugated secondary antibodies,
   including goat anti-mouse IgG conjugated with Alexa Fluor 488 or 647,
   or goat anti-rabbit IgG labeled with Alexa Fluor 405 (Jackson
   ImmunoResearch). Images were captured using a Nikon Eclipse
   fluorescence microscope and thePANNORAMIC, MIDI digital slide scanner
   (manufacturer: 3DHISTECH). Primary antibody used Immunohistochemistry
   and immunofluorescence including KIM1(R&D system, AF1817),
   LC3B(novusbio, NB100-2220), PDGFRB(ABclonal, A19531), α-SMA(Cell
   Signaling, 19245), FGF2(ABclonal,A11488), pS6K (ABclonal,A4898; Cell
   Signaling #9234), Ki67(Cell Signaling,9129T), ATG7(proteintech,
   10088-2-AP), LARS1 (proteintech 21146-1-AP) Slc7a8(Thermo Fisher
   Scientific,ANT-108-200UL), Slc7a5(Thermo Fisher Scientific,
   PA5-115916), mTORC1 (SANTA CRUZ, sc-517464).Fibrosis was measured by
   randomly selecting 10 fields of view for each mouse kidney under a 200x
   magnification and then calculating the percentage of fibrotic area
   (collagen). The ImageJ tool was used to calculate the collagen volume
   fraction, that is, the percentage of the blue area (collagen) to the
   total area of each field.

2.6. Western blot analysis

   Kidney tissues or cultured renal tubular cells were lysed in RIPA
   buffer containing protease and phosphatase inhibitors. Protein
   concentrations were determined using a BCA protein assay kit
   (Beyotime). Equal amounts of protein (30–50 μg) were separated by
   SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with
   5 % BSA and incubated overnight at 4 °C with primary antibodies against
   pS6K1 (Cell Signaling Technology, Thr389, 9205), S6K1 (Cell Signaling
   Technology, 9202), LARS1 (Bethyl Laboratories and proteintech
   21146-1-AP), ULK1 (Cell Signaling Technology,8054T), p-ULK1 (Ser757)
   (Cell Signaling Technology, 6888), p62 (proteintech,18420-1-AP), FGF2
   (ABclonal,A11488), mTORC1 (SANTA CRUZ, sc-517464), p-mTOR (ABclonal,
   AP0115), CTGT (ABclonal, A11067), PDGFRB (ABclonal, A19531), α-SAM
   (Cell Signaling, 19245), α-Tububin (Proteintech, 11224-1-AP), and GAPDH
   (Servicebio,GB15004-100). After washing, membranes were incubated with
   HRP-conjugated secondary antibodies and developed using an ECL
   detection system. Densitometric analysis was performed using ImageJ
   software.

2.7. Metabolomic analysis

   Metabolites were extracted by homogenizing 60 mg of tissue with water
   and a methanol-acetonitrile solution containing internal standards,
   followed by protein precipitation at −20 °C and centrifugation at
   14,000 rcf for 20 min at 4 °C. Chromatographic separation was performed
   on an Agilent 1290 UHPLC system using a gradient of ammonium acetate
   and acetonitrile solutions. Mass spectrometric analysis was conducted
   on a 6500/5500 QTRAP system with electrospray ionization in positive
   mode, employing multiple reaction monitoring (MRM) for quantification.
   Data were analyzed using MultiQuant 3.0.2, with internal standards for
   retention time correction and normalization.

2.8. Cell culture and treatment

   The mouse proximal tubular cells (mPTCs) were originally obtained from
   Sciencell Research Laboratories and cultured in DMEM medium
   supplemented with 10 % fetal bovine serum and growth factors. Small
   interference RNAs (siRNAs) were synthesized by Sangon Biotech
   (Shanghai) Co., Ltd. Small interfering RNAs were transfected by
   Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific; 13778030),
   according to the manufacturer's protocol. A non-silencing siRNA
   oligonucleotide that does not recognize any known homolog of mammalian
   genes was used as a negative control.

2.9. Glucose (Glu) or branched-chain amino acids (BCAAs) starvation and
stimulation protocol

   For glucose deprivation, cells were washed twice with glucose-free DMEM
   and then incubated in glucose-free DMEM. Stimulation was performed by
   adding DMEM containing 4.5 mg/ml glucose for 5–30 min. Each gram of
   BCAAs (Solarbio) supplement contains 0.2 g of isoleucine, 0.4 g of
   leucine, and 0.2 g of valine. For BCAAs starvation, cells were washed
   and incubated in BCAAs-free DMEM for 50 min, followed by stimulation
   with BCAAs-free DMEM supplemented with 800 μM BCAAs for 10 min.

2.10. Co-immunoprecipitation (Co-IP)

   Renal tubular cells were lysed in IP lysis buffer (Beyotime) containing
   protease and phosphatase inhibitors. Cell lysates (500 μg of protein)
   were pre-cleared with Protein A/G agarose beads and incubated with
   anti-Flag or anti-ULK1 antibodies overnight at 4 °C. Immune complexes
   were captured with Protein A/G beads, washed, and eluted in SDS sample
   buffer. Co-immunoprecipitated proteins were analyzed by Western
   blotting as described above.

2.11. RNA sequencing and Gene Ontology pathway analyses

   Total RNA extraction from renal cortex was performed with Trizol
   reagent (Invitrogen, USA), according to the manufacturer's
   instructions. The RNA concentration was calculated by a NanoDrop
   spectrophotometer. RNA sequencing analysis was performed by BGI
   (Beijing Genomics Institute) (Shenzhen, China). The differentially
   expressed genes were analyzed using DESeq2. The functional annotation
   tool at [64]https://geneontology.org was used to conduct Gene Ontology
   (GO) pathway analysis. Additionally, the DAVID 6.8 functional
   annotation tool ([65]https://david.ncifcrf.gov) was employed to perform
   KEGG pathway analysis, focusing on the identification of the top 20
   enriched pathways.

2.12. Statistical analysis

   All data are presented as mean ± SEM. Statistical significance was
   determined using one-way ANOVA followed by Tukey's post hoc test or
   two-way ANOVA as appropriate. P < 0.05 was considered statistically
   significant. Statistical analyses were performed using GraphPad Prism
   version 8.0.

3. Results

3.1. CR exacerbates renal injury and fibrosis following IRI

   We first assessed the impact of CR on renal injury following unilateral
   IRI ([66]Fig. 1A). Mice subjected to CR after IRI (IRI-14d + CR)
   exhibited significantly higher levels of serum CRE and BUN compared to
   those in the Non-CR group, indicating exacerbated renal dysfunction
   ([67]Fig. 1B and C). Immunohistochemical analysis showed elevated
   expression of markers associated with fibrosis and injury, including
   KIM1, PDGFRB, and α-SMA ([68]Fig. 1D). Histological analysis further
   revealed increased tubular injury in the CR group, with elevated
   tubular injury scores ([69]Fig. 1E). Additionally, we confirmed via
   immunoblot that FN, α-SMA, PDGFRB, and CTGF were significantly
   upregulated in the post-surgery CR-treated group ([70]Fig. 1F and G).
   These findings suggest that CR worsens renal outcomes following IRI.

Fig. 1.

   [71]Fig. 1
   [72]Open in a new tab

   CR exacerbates renal function impairment and fibrosis induced by IRI.
   (A) Schematic representation of the experimental design. Mice underwent
   unilateral ischemia-reperfusion injury (28 min) followed by CR or NonPC
   for 14 days. Uninephrectomy was performed on day 13, and mice were
   sacrificed on day 14 for analysis. (B) Gross morphology of the kidneys
   from sham-operated and IRI-14d mice under NonPC and CR conditions,
   showing differences in kidney size and appearance. Scale bars: 1 cm.
   (C) CRE and BUN levels in sham and IRI-14d mice under NonPC and CR
   conditions. Data are presented as mean ± SEM. n = 8 per group.
   ∗P < 0.05 versus Sham; #P < 0.05 versus NonPC. (D) Representative
   images of kidney sections stained with H&E, KIM1, VIM, and PDGFRB in
   sham and IRI-14d mice under NonPC and CR conditions. Scale bars: 50 μm.
   Red arrows in the H&E panels indicate areas of tubular injury. (E)
   Quantification of tubular injury score in sham and IRI-14d mice under
   NonPC and CR conditions. Data are presented as mean ± SEM. n = 8 per
   group. ∗P < 0.05 versus Sham; #P < 0.05 versus NonPC. (F) Western blot
   analysis of fibronectin (FN), α-SMA, PDGFRB, and CTGF in kidney tissues
   from sham and IRI-14d mice under NonPC and CR conditions. (G)
   Quantification of protein expression levels of FN, α-SMA, PDGFRB, and
   CTGF from Western blots. Data are presented as mean ± SEM. n = 8 per
   group. ∗P < 0.05 versus Sham; #P < 0.05 versus NonPC.

3.2. CR influences mTORC1 signaling and autophagy activation in the injured
kidney

   Subsequently, to elucidate the molecular mechanisms underlying the
   deleterious effects of CR, we performed transcriptomic analysis and
   identified a set of differentially expressed genes (Supplemental
   Datasets 1). Enrichment analysis of these genes indicated that CR
   predominantly affects the mTORC1 signaling pathway, autophagy, and
   fibrosis within the kidneys ([73]Fig. 2A and B). Immunoblot analysis
   revealed that CR suppressed mTORC1 signaling, as indicated by decreased
   phosphorylation of mTORC1 and its downstream target pS6K in CR-treated
   IRI mice ([74]Fig. 2C and D). Consistent with this, immunofluorescence
   analysis demonstrated reduced pS6k expression in proximal tubules of
   CR-treated mice, along with lower Ki67 positive tubular cells,
   indicating decreased cell proliferation ([75]Fig. 2E).

Fig. 2.

   [76]Fig. 2
   [77]Open in a new tab

   CR affects mTORC1 signaling in the kidney following IRI. (A) Volcano
   plot showing differentially expressed genes in the kidneys of IRI-14d
   mice under NonPC versus CR conditions. Genes upregulated in CR are
   indicated in red, and downregulated genes are indicated in black. (B)
   Pathway enrichment analysis of the differentially expressed genes. (C)
   Western blot analysis of p-mTOR, total mTOR, and pS6k in kidney tissues
   from sham and IRI-14d mice under NonPC and CR conditions. GAPDH was
   used as a loading control. (D) Quantification of p-mTOR/mTOR and pS6K
   expression levels from Western blots. Data are presented as mean ± SEM.
   n = 8 per group. ∗P < 0.05 versus Sham; #P < 0.05 versus NonPC. (E)
   Representative immunofluorescence images of pS6k (red) and Lotus
   tetragonolobus lectin (LTL, green) staining, and Ki67 (red) and DAPI
   (blue) staining in kidney tissues from sham and IRI-14d mice under
   NonPC and CR conditions. Scale bars: 20 μm. Insets show higher
   magnification images of the boxed regions.

   To further investigate the inhibitory effect of CR on the mTORC1
   signaling pathway, we employed renal injection of adenoviruses to
   overexpress S6K ([78]SFig. 1A). Mice subjected to unilateral IRI
   (28 min) were injected of adenoviruses encoding either a Ad-NC or
   Ad-S6K and treated with unilateral nephrectomy on day 13. The results
   indicated that overexpression of pS6K significantly improved renal
   function ([79]SFig. 1B). Immunoblot analysis confirmed a marked
   increase in S6K expression in the kidneys of mice treated with Ad-S6K
   ([80]SFig. 1C and D). Histological examination revealed reduced tubular
   damage and interstitial fibrosis in the kidneys of mice overexpressing
   S6K ([81]SFig. 1E). Quantitative analysis of tubular damage scores and
   interstitial fibrosis percentage further supported these findings
   ([82]SFig. 1F and G). These results suggest that overexpression of S6K
   activates the mTORC1 pathway and alleviates CR-induced renal fibrosis
   and dysfunction following IRI.

   We also found that CR significantly enhanced autophagy activation, as
   indicated by the transcriptomic data showing increased expression of
   autophagy-related genes (Map1lc3b, Ulk1, and Atg9a) in renal tissue
   ([83]Fig. 3A). Immunoblot analysis further confirmed this,
   demonstrating an increase in LC3 II/I levels and a decrease in p62
   levels ([84]Fig. 3B and C). Immunohistochemical staining also
   corroborated these findings, showing more pronounced LC3B and
   ULK1-positive areas in the kidneys of CR-treated mice ([85]Fig. 3D). To
   examine the location, dynamics, and flux of autophagy, we employed
   autophagy reporter mice expressing a tandem RFP-GFP-LC3 fusion protein.
   This fusion protein emits both GFP and RFP fluorescence in
   autophagosomes under neutral pH conditions, but the GFP signal is lost
   as autophagosomes mature into autolysosomes in an acidic environment
   [[86]20,[87]25]. As shown in [88]Fig. 3E, sham-operated control mice
   exhibited low levels of autophagy in renal tubules, with minimal
   GFP-LC3 and RFP-LC3 puncta. However, following unilateral renal IRI,
   there was a marked increase in the formation of autophagosomes (yellow
   puncta representing overlapping GFP-LC3 and RFP-LC3) and autolysosomes
   (red-only RFP-LC3 puncta) in renal tubules at all CR treatment time
   points ([89]Fig. 3E). Furthermore, autophagic flux was significantly
   elevated in CR-treated mice ([90]Fig. 3F and G). These results indicate
   that CR inhibits the mTORC1 signaling pathway, enhances autophagy, and
   contributes to the exacerbation of renal injury.

Fig. 3.

   [91]Fig. 3
   [92]Open in a new tab

   CR exacerbates autophagy activation following IRI. (A) Heatmap showing
   the expression levels of key autophagy-related genes (Map1lc3b, Ulk1,
   Atg9a) in kidneys from IRI-14d mice under NonPC and CR conditions. (B)
   Western blot analysis of p-Ulk1, total Ulk1, LC3B, and P62 in kidney
   tissues from sham and IRI-14d mice under NonPC and CR conditions. GAPDH
   was used as a loading control. (C) Quantification of p-Ulk1/Ulk1 and
   P62 expression levels from Western blots. Data are presented as
   mean ± SEM. n = 8 per group. ∗P < 0.05 versus Sham; #P < 0.05 versus
   NonPC. (D) Representative immunohistochemical images showing Ulk1 and
   LC3B staining in kidney tissues from sham and IRI-14d mice under NonPC
   and CR conditions. Scale bars: 100 μm. (E) Representative
   immunofluorescence images of autophagosomes (RFP, red) and
   autolysosomes (GFP, green) in kidney tissues from sham and IRI-14d mice
   under NonPC and CR conditions. Nuclei are stained with DAPI (blue).
   Insets show higher magnification images of the boxed regions. Scale
   bars: 50 μm. (F) Quantification of LC3 puncta (autophagosomes and
   autolysosomes) per proximal tubule in the different groups. Data are
   presented as mean ± SEM. n = 8 per group. ∗P < 0.05 versus Sham. (G)
   Autophagic flux rate quantified based on the ratio of autolysosomes to
   autophagosomes in the different groups. Data are presented as
   mean ± SEM. n = 8 per group. ∗P < 0.05 versus Sham.

3.3. Rapamycin mimics the effects of CR on renal injury and autophagy
activation

   Given that rapamycin is a specific inhibitor of mTOR, we examined
   whether it could mimic the effects of CR following IRI ([93]Fig. 4A).
   Mice treated with rapamycin after IRI exhibited elevated serum CRE and
   BUN levels ([94]Fig. 4B). The transcriptomic profiles in the middle
   panel (IRI-14d + CR vs IRI-14d + Rap) indicate substantial overlap
   between CR and rapamycin treatment, suggesting that rapamycin might
   replicate the effects of CR in the context of IRI through shared
   molecular pathways ([95]Fig. 4C, Supplemental Datasets 2 and 3).
   Immunoblot and immunohistochemical analyses confirmed that rapamycin
   enhanced autophagy activation, as indicated by increased LC3 II/I
   levels and decreased p62 expression ([96]Fig. 4D). Additionally, mice
   treated with rapamycin after IRI showed increased renal injury and
   fibrosis ([97]Fig. 4E), elevated tubular injury scores([98]Fig. 4F),
   and increased interstitial fibrosis ([99]Fig. 4G). These findings
   support the notion that CR's effects on renal injury are mediated, at
   least in part, through mTORC1 inhibition and enhanced autophagy.

Fig. 4.

   [100]Fig. 4
   [101]Open in a new tab

   Rapamycin mimics the effects of CR on renal injury following IRI. (A)
   Schematic of the experimental design. Mice underwent unilateral IRI
   (28 min) followed by uninephrectomy and treatment with either saline or
   Rap (2.5 mg/kg/day) for 14 days. (B) CRE and BUN levels in sham and
   IRI-14d mice treated with saline or Rap. Data are presented as
   mean ± SEM. n = 8 per group. ∗P < 0.05 versus Sham; #P < 0.05 versus
   Saline. (C) Volcano plots showing differentially expressed genes in the
   kidneys of IRI-14d mice under the indicated conditions. Upregulated
   genes are shown in red, and downregulated genes are shown in black. (D)
   Western blot analysis of LC3B in kidney tissues from sham and IRI-14d
   mice treated with saline or Rap. GAPDH was used as a loading control.
   Quantification of LC3BⅡ/Ι expression levels is shown to the right. Data
   are presented as mean ± SEM. n = 8 per group. ∗P < 0.05 versus Sham;
   #P < 0.05 versus Saline. (E) Representative images of kidney sections
   stained with H&E, KIM1, α-SMA, and Masson's trichrome in sham and
   IRI-14d mice treated with saline or rapamycin. Scale bars: 100 μm. (F)
   Quantification of tubular injury score in the different groups. Data
   are presented as mean ± SEM. n = 8 per group. ∗P < 0.05 versus Sham;
   #P < 0.05 versus Saline. (G) Quantification of interstitial fibrosis
   percentage in the different groups, as assessed by Masson's trichrome
   staining. Data are presented as mean ± SEM. n = 8 per group. ∗P < 0.05
   versus Sham; #P < 0.05 versus Saline.

3.4. Both Atg7 deficiency and autophagy inhibition through 3-MA can mitigate
the detrimental effects of CR

   Atg7 is critical for the autophagy process, particularly in
   facilitating conjugation events [[102]26]. To elucidate the role of
   tubular autophagy and CR in kidney repair after IRI, we generated an
   inducible, renal tubule-specific Atg7 knockout mouse model by breeding
   Pax8-rtTA^±/tetO^± mice [[103]27,[104]28] with Atg7^flox/flox mice to
   produce iRT-atg7-KO mice and wild type (iRT-Atg7- WT) littermates
   ([105]Fig. 5A). The expression of ATG7 was deleted in the kidneys of
   doxycycline-induced iRT-Atg7-KO mice ([106]Fig. 5B). Immunofluorescence
   co-localization confirmed the loss of Atg7 in the proximal tubules of
   iPT-Atg7-KO mice ([107]Fig. 5C). In contrast to wild-type (WT) mice,
   Atg7-deficient mice exhibited significantly reduced renal injury and
   fibrosis after CR, as shown by lower serum CRE and BUN levels
   ([108]Fig. 5D). Histological analysis demonstrated that Atg7 deletion
   in CR-treated IRI mice resulted in reduced tubular injury and
   interstitial fibrosis, evidenced by lower injury scores and reduced
   fibrosis ([109]Fig. 5E–G). In addition, mice subjected to unilateral
   IRI (28 min) were treated with either saline or 3-MA, with or without
   CR, for 14 days ([110]SFig. 2A). Results indicated that under CR
   conditions, 3-MA treatment significantly reduced serum CRE and BUN
   levels ([111]SFig. 2B). Immunoblot analysis demonstrated that 3-MA
   effectively inhibited excessive autophagy activation ([112]SFig. 2C).
   Additionally, tubular injury scores further confirmed that 3-MA
   treatment alleviated tubular damage ([113]SFig. 2D). Histological
   examination of kidney tissues from the 3-MA treatment group, using H&E,
   KIM1, Masson's trichrome, and LC3B staining, showed a significant
   reduction in interstitial fibrosis and autophagy activation ([114]SFig.
   2E and F). These results suggest that autophagy plays a critical role
   in mediating CR-induced renal injury. Furthermore, previous studies
   have demonstrated that following AKI, fibrotic tubular cells produce
   FGF2 via autophagy, which contributes to fibroblast activation and
   kidney fibrosis [[115]29]. Immunoblot analysis showed a significant
   increase in FGF2 levels in WT kidneys two weeks after unilateral
   ischemic AKI, whereas this increase was substantially lower in
   iRT-Atg7-KO mice ([116]Fig. 5H). To further examine FGF2 expression and
   localization in the kidney, we performed immunohistochemical staining
   ([117]Fig. 5I). FGF2 was rarely detected in sham control kidneys. After
   ischemic AKI, FGF2 accumulated in the cytoplasm of atrophic tubules,
   primarily on the basolateral side, in WT kidneys. In contrast, this
   tubular expression of FGF2 was significantly suppressed in iRT-Atg7-KO
   mice ([118]Fig. 5I).

Fig. 5.

   [119]Fig. 5
   [120]Open in a new tab

   The tubule autophagy defect in iRT-atg7-KO mice inhibits the effect of
   CR on kidney fibrosis following ischemia. (A) Breeding strategy for
   generating inducible, renal tubule-specific Atg7 knockout mice. (B) At
   the specified time points after treatment with doxycycline, proteins
   were extracted from the iPT-Atg7-WT and iRT-atg7-KO kidneys, and ATG7
   expression was detected by Western blot. (C) Representative
   immunofluorescence images of ATG7 (red) and Lotus tetragonolobus lectin
   (LTL, green) staining, along with DAPI (blue) staining, in renal tissue
   of iPT-Atg7-KO and WT mice. (D) Serum CRE and BUN levels of iPT-Atg7-KO
   or WT mice treated with IRI-14d under NonPC and CR conditions. Data are
   presented as mean ± SEM. n = 8 per group. ∗P < 0.05 versus NonPC; ns,
   not significant. (E) Representative images of kidney sections stained
   with H&E, Masson, Sirius Red and PDGFRB's trichrome from iPT-Atg7-KO
   and WT mice under NonPC and CR conditions. Scale bars: 100 μm. (F)
   Quantification of tubular injury score in the different groups. Data
   are presented as mean ± SEM. n = 8 per group. ∗P < 0.05 versus NonPC;
   ns, not significant. (G) Quantification of interstitial fibrosis
   percentage in the different groups, as assessed by Masson's trichrome
   staining. Data are presented as mean ± SEM. n = 8 per group. ∗P < 0.05
   versus NonPC; ns, not significant. (H) Western blot analysis of FGF2 in
   kidney tissues from sham and IRI-14d mice under NonPC and CR
   conditions. GAPDH was used as a loading control. (I) FGF2 IHC. Scale
   bar: 100 μm. The right bar graph shows the quantitative analysis of the
   FGF2 positive staining area. Data are presented as mean ± SEM. n = 8
   per group. ∗P < 0.05 versus Sham; #P < 0.05 versus iPT-Atg7-WT.

3.5. Supplementation with glucose but not BCAAs alleviated CR-induced renal
fibrosis and dysfunction following IRI

   mTORC1, a key regulator of nutrient sensing, plays a central role in
   cellular metabolism and stress responses. We investigated the effects
   of nutrient supplementation, including BCAAs, aspartate (Asp),
   asparagine (Asn), and Glu, to activate mTOR and evaluate their roles in
   this model of kidney injury and repair ([121]Fig. 6A). We performed
   targeted metabolomics analysis to examine changes in amino acids and
   their derivatives in kidney tissues across different groups
   (Supplemental Datasets 4, 5, and 6), revealing significant differences
   between mice treated with CR and without CR following IRI ([122]Fig. 6B
   and C). Interestingly, Glucose supplementation resulted in a
   significant reduction in serum CRE and BUN levels compared to the
   non-supplemented CR-treated mice, whereas supplementation with various
   amino acids did not produce similar effects ([123]Fig. 6D).
   Histopathological analysis further demonstrated that only glucose
   supplementation led to a reduction in tubular injury and interstitial
   fibrosis ([124]Fig. 6E–G). Additionally, immunohistochemical staining
   revealed a significant increase in pS6k expression in the
   glucose-supplemented group ([125]Fig. 6E). Western blot analysis
   corroborated these findings, showing that glucose supplementation
   reactivated the mTORC1 pathway (pS6k) ([126]Fig. 6H). These results
   indicate that it is the supplementation of glucose, rather than amino
   acids, that can alleviate the exacerbation of renal fibrosis and
   dysfunction induced by CR.

Fig. 6.

   [127]Fig. 6
   [128]Open in a new tab

   The supplementation of glucose, rather than BCAAs, can mitigate the
   exacerbation of CR-affected renal fibrosis and dysfunction. (A)
   Schematic of the experimental design. Mice underwent unilateral IRI
   (28 min) followed by CR with or without supplementation of BCAAs, Asp,
   Asn, or Glu for 14 days. (B) Heatmap showing the levels of various
   metabolites in the kidneys. (C) Volcano plots showing differentially
   expressed metabolites in the kidneys of IRI + CR mice with or without
   supplementation with BCAAs, Asp, Asn, or Glu. Upregulated metabolites
   are shown in red, and downregulated metabolites are shown in blue. (D)
   Serum CRE and BUN levels in the different treatment groups. Data are
   presented as mean ± SEM. n = 8 per group. ∗P < 0.05 versus NC. (E)
   Representative images of kidney sections stained with H&E, Masson's
   trichrome, PDGFRB, and pS6K in the different treatment groups. Red
   arrowheads in the pS6K panels indicate positive staining. Scale bars:
   100 μm. (F) Quantification of tubular injury scores in the different
   groups. Data are presented as mean ± SEM. n = 8 per group. ∗P < 0.05
   versus NC. (G) Quantification of interstitial fibrosis percentage in
   the different groups, as assessed by Masson's trichrome staining. Data
   are presented as mean ± SEM. n = 8 per group. ∗P < 0.05 versus NC. (H)
   Immunoblot analysis of pS6K, S6K, and GAPDH in kidney tissues from the
   different groups. Numbers represent quantification of band intensities
   relative to GAPDH.

   To elucidate this phenomenon, we performed a metabolomic analysis of
   kidney tissues from the model. We were surprised to find that the
   levels of isoleucine, leucine, and valine were unexpectedly elevated in
   the kidneys of mice subjected to CR, with isoleucine and leucine being
   particularly pronounced ([129]SFig. 3A). This finding may explain why
   amino acid supplementation did not improve the adverse effects caused
   by CR. Further analysis of transcriptomic data from kidney tissues
   revealed a decrease in genes related to amino acid catabolism
   ([130]Sfig. 3B) and a dysregulation in the expression of
   transporter-related genes ([131]SFig. 3B). Immunofluorescence analysis
   further demonstrated that CR disrupted amino acid transport, as
   evidenced by altered expression levels of the essential amino acid
   transporters Slc7a8 and Slc7a5. CR appears to exacerbate abnormalities
   in BCAAs catabolism following IRI, leading to elevated renal BCAAs
   levels, indicating that the reduction in mTORC1 activity was not caused
   by decreased BCAAs levels.

3.6. Glucose is indispensable for the activation of mTORC1 in renal tubular
cells

   To investigate the glucose and amino acid requirements for mTORC1
   activation, we assessed the effects of their supplementation on the
   mTORC1 signaling pathway in renal tubular cells. Western blot analysis
   revealed that mTORC1 activation by BCAAs was glucose-dependent
   ([132]Fig. 7A). Different amino acids can activate Rag guanosine
   triphosphatase through their corresponding aminoacyl-tRNA synthetases,
   thereby activating mTORC1 [[133]30]. By silencing the branched-chain
   amino acid sensors LARS1, IARS1, and VARS1, we found that LARS1
   knockdown was most critical for mTORC1 activation, as its loss
   abrogated nutrient-induced mTORC1 signaling ([134]Fig. 7B). In the
   presence of glucose and leucine, LARS1 translocates to the lysosome and
   facilitates the conversion of RagD-GTP to RagD-GDP, leading to mTORC1
   activation [[135]31]. Immunofluorescence confirmed the colocalization
   of LARS1 with mTORC1 in the presence of glucose, emphasizing the
   importance of glucose in mTORC1 activation ([136]Fig. 7C and D). We
   downloaded 3D protein structures of LARS1 and RagD from the RCSB
   database ([137]https://www.rcsb.org/). Using Discovery Studio and
   Ligplus software, we performed 3D and 2D force analysis and
   visualization ([138]Fig. 7E). Co-IP experiments revealed an enhanced
   interaction between overexpressed and tagged LARS1 and RagD under
   glucose-rich conditions. This interaction was reduced upon glucose
   deprivation but was partially restored in the add-back condition
   ([139]Fig. 7F). These results suggest that the interaction between
   LARS1 and RagD is sensitive to glucose availability.

Fig. 7.

   [140]Fig. 7
   [141]Open in a new tab

   Activation of mTORC1 in renal tubular cells requires the presence of
   both glucose and amino acids. (A) Immunoblot analysis showing the
   effects of BCAAs and Glu on the p-S6K in renal tubular cells. GAPDH is
   shown as a loading control. (B) Western blot analysis of p-S6K, S6K,
   and the expression of LARS1, IARS1, and VARS1 in renal tubular cells
   transfected with control siRNA (si-con) or si-RNAs targeting LARS1,
   IARS1, or VARS1, with or without glucose. GAPDH is shown as a loading
   control. (C) Representative immunofluorescence images showing the
   colocalization of LARS1 (blue), Lysotracker (red), and mTORC1 (green)
   in renal tubular cells, with or without Glu. Scale bars: 20 μm. (D) The
   bar graph quantifies the percentage of co-localized pixels in (C)
   compared to the control. Data are presented as mean ± SEM. ∗P < 0.01
   versus -Glu group. (E) Molecular docking was used to verify the binding
   activity between RagD and LARS1 proteins. The red ribbon structure
   represents the RagD protein, while the green ribbon structure
   represents the LARS1 protein. (F) Co-IP analysis showing the
   interaction between LARS1 and RagB/RagD in renal tubular cells, with or
   without glucose, in the presence of amino acids. −/+,add back Glu.

3.7. ULK1 phosphorylates LARS1, inhibiting mTORC1 activation in the absence
of glucose

   Finally, we explored the role of ULK1, a key regulator of autophagy, in
   modulating mTORC1 activation under nutrient-deprived conditions. LARS1
   was identified as possible ULK1-interacting protein [[142]32] and
   potential member of an autophagy network by mass spectrometry
   [[143]33]. Co-immunoprecipitation experiments revealed that in the
   absence of glucose, ULK1 phosphorylates LARS1, leading to its
   inactivation and preventing mTORC1 activation ([144]Fig. 8A). The
   association of overexpressed and tagged LARS1 and ULK1 was increased in
   cells deprived of glucose ([145]Fig. 8B). These results suggest that
   ULK1-mediated inactivation of LARS1 underlies the failure of mTORC1
   activation in CR conditions. Upon sensing leucine in the presence of
   glucose, LARS1 supports translation by leucylating tRNA and also by
   activating mTORC1. However, in the absence of glucose, LARS1
   phosphorylated by ULK1 loses its leucine binding capability, decreasing
   both activities, thus leading to the inhibition of protein synthesis
   and activation of autophagy ([146]Fig. 8C).

Fig. 8.

   [147]Fig. 8
   [148]Open in a new tab

   In the absence of glucose, ULK1 phosphorylates LARS1, reducing mTORC1
   activation. (A) Co-IP analysis showing the interaction between LARS1
   and ULK1 in renal tubular cells in the presence or absence of Glu.
   Western blot analysis was performed using anti-ULK1 and anti-LARS1
   antibodies. IgG serves as a control (B) Western blot analysis of
   ULK1,p-S6K, S6K, and LARS1 in renal tubular cells transfected with
   Flag-LARS1, with or without Glu for the indicated times, in the
   presence of amino acids. GAPDH is shown as a loading control. (C)
   Schematic diagram of caloric restriction modulates mTORC1 signaling to
   influence autophagy, fibrosis, and maladaptation.

4. Discussion

   In this study, we explored the impact of CR on renal injury and
   fibrosis following IRI, revealing that CR exacerbates renal
   dysfunction, fibrosis, and autophagy activation. Although CR has shown
   protective effects in various acute and chronic kidney diseases, these
   findings highlight a previously unappreciated detrimental effect of CR
   on renal tubule repair in the context of recovery after AKI.

   The beneficial effects of CR on health are believed to stem from
   optimized cellular energy utilization and the induction of adaptive
   cellular stress responses [[149]10]. Previous studies have shown that
   in the gut, fasting and refeeding can enhance intestinal stem cell
   function and intestinal epithelial repair [[150]34], while in muscle,
   fasting induces muscle stem cells to enter a deep quiescent state,
   delaying muscle regeneration after injury [[151]35]. These observations
   suggest that the effects of CR on various tissues and stem cell systems
   may be context-dependent. After kidney injury, repair may result in the
   formation of a disorganized cellular and extracellular matrix, or it
   can regenerate the original tissue structure through a regenerative
   process. The repair of the existing nephron units after tubular damage
   is an evolutionarily conserved process [[152]36]. CR triggers
   evolutionarily conserved adaptive responses, halting renal tissue
   repair to conserve resources and attempting to promote adaptation and
   survival, with mTOR playing a key role in this process, as revealed in
   this study.

   mTORC1, a central regulator of cell growth and metabolism, integrates
   signals from nutrients, energy status, and stress [[153]37]. However,
   the effects of mTORC1 inhibition can be context-dependent, with studies
   indicating both protective and potentially harmful effects in different
   disease models. For example, recent research has shown that CR during
   pregnancy affects mTORC1 signaling, reducing nephron progenitor cell
   proliferation in offspring, which may impair kidney function later in
   life [[154]38]. Notably, supplementation with methionine or increasing
   mTORC1 activity during pregnancy reversed the negative effects of CR on
   nephron progenitor cell proliferation. Our results demonstrate that CR
   and mTORC1 inhibition significantly worsens renal outcomes after IRI,
   as evidenced by renal function impairment, and exacerbated tubular
   injury and interstitial fibrosis. mTORC1 is activated by growth
   factors, nutrients, and cellular energy status, while under
   energy-deficient conditions, its activity is downregulated to limit
   cellular growth. In the injured kidney, mTORC1 activation under
   nutrient-rich conditions promotes tubular cell proliferation and
   recovery [[155]14]. However, under nutrient-deficient conditions, the
   injured kidney becomes a victim of the systemic downregulation of
   mTORC1 activity, leading to impaired renal repair and exacerbated
   fibrosis. Forced overexpression of S6K1 in the injured kidney to
   activate the mTORC1-S6K1 downstream pathway could potentially balance
   systemic nutrient supply, rescue the damaged kidney, and mitigate the
   detrimental effects associated with CR.

   Rapamycin with immunosuppressive and antitumor effects, is widely used
   in combination with calcineurin inhibitors to enhance immunosuppression
   while mitigating their side effects [[156]39]. IRI is a major cause of
   DGF [[157]23], and the use of rapamycin in DGF patients provides a
   valuable clinical model to observe the effects of mTOR inhibition on
   renal repair. A comprehensive literature search was conducted using
   PubMed, Embase, Scopus, Cochrane Library, Web of Science, Ovid, and
   ProQuest, identifying five studies [[158][40], [159][41], [160][42],
   [161][43], [162][44]] that reported the effect of rapamycin on the
   duration of DGF in patients after renal transplantation. These studies
   demonstrated longer DGF durations in rapamycin-treated groups compared
   to controls, although not all studies reported statistically
   significant differences. Specifically, in Kelly D. Smith et al.
   reported the mean DGF duration was 27.6 days with rapamycin versus 6.4
   days without rapamycin [[163]41]. Laetitia Albano et al. demonstrated
   that the values were 10.2 days and 7.6 days, respectively [[164]44].
   Similarly, Ryan A. McTaggart et al. reported a mean duration of 14.86
   days in the rapamycin group versus 9.59 days in the control group
   [[165]42]. Stallone Giovanni et al. showed the DGF duration was 6.2
   days with rapamycin and 3.2 days without rapamycin [[166]40]. Finally,
   W. Tahira 2015 observed a substantial increase in DGF duration in the
   rapamycin group (42.3 days) compared to the control group (26.5 days)
   [[167]43]. This suggests that the mechanism by which mTOR inhibition
   impedes post-ischemic kidney repair may be a common feature in humans.

   CR inhibits mTORC1 activity, thereby reducing cellular growth signals
   and activating autophagy—a process that is generally protective by
   clearing damaged organelles and proteins [[168]19]. The link between
   CR, autophagy, and mTORC1 inhibition provides a potential mechanistic
   explanation for the observed renal damage and renal fibrosis. We
   demonstrated that the autophagy inhibitor 3-MA and Atg7 gene knockout
   mitigated CR-induced renal injury, suggesting that the exacerbation of
   renal damage by CR was at least partially mediated through autophagy.
   This finding aligns with previous studies showing that dysregulated
   autophagy can contribute to the progression of renal fibrosis and
   tubular injury by paracrine secretion of pro-fibrotic cytokines
   [[169]45]. By inhibiting autophagy, tubular Atg7 ablation reduced
   fibrosis by decreasing the pro-fibrotic factor FGF2, supporting the
   hypothesis that excessive autophagy plays a crucial role in caloric
   restriction-induced renal damage. Interestingly, rapamycin mimicked the
   effects of CR on renal injury and autophagy activation, further
   corroborating the involvement of the mTORC1-autophagy axis in mediating
   the effects of CR. This highlights the complexity of mTORC1 signaling
   in kidney disease, where its inhibition can have both protective and
   detrimental effects depending on the context and extent of autophagy
   activation.

   Given that BCAAs are critical for mTORC1 activation, their depletion
   likely contributes to the impaired mTORC1 signaling observed in
   CR-treated mice. Supplementation with BCAAs may potentially benefit
   kidney recovery. Interestingly, targeted metabolomics detected an
   increase, rather than a decrease, in BCAAs. This may reflect a
   reduction in amino acid catabolism, enhanced absorption capacity, and
   the production of amino acids via cellular autophagy. This also
   explains why exogenous supplementation of BCAAs indeed increased the
   levels of BCAAs in the kidneys, but did not benefit kidney repair. We
   noticed that CR significantly reduced fasting blood glucose, so the
   inhibition of mTORC1 may be attributed to the decrease in blood
   glucose. Interestingly, glucose supplementation improved fibrosis and
   reduced excessive autophagy in the kidneys. This finding is consistent
   with the notion that both glucose and amino acid availability are
   essential for mTORC1 activity [[170]46], and their disruption can have
   significant consequences for cellular function and survival [[171]47].
   Finally, we explored the mechanism by which glucose regulates mTORC1.
   Our study also identified a key role for ULK1, a regulator of
   autophagy, in modulating LARS1 activity and mTORC1 signaling. We found
   that in the absence of glucose, ULK1 phosphorylates LARS1, leading to
   its inactivation and subsequent failure to activate mTORC1. This
   mechanism likely contributes to the inability of mTORC1 to be activated
   under CR conditions, further exacerbating renal injury. The interplay
   between nutrient availability, ULK1 activity, and mTORC1 signaling
   underscores the complexity of metabolic regulation in the kidney and
   its impact on disease outcomes.

   Overall, our findings suggest that CR application following IRI, may be
   detrimental due to its effects on mTORC1 signaling and autophagy. The
   potential therapeutic implications of targeting the mTORC1-autophagy
   axis in kidney disease warrant further investigation, particularly in
   the condition of tissue regeneration, such as recovery after AKI.
   Future studies should explore the long-term effects of CR in different
   models of kidney injury and identify potential strategies to modulate
   autophagy and mTORC1 signaling to optimize renal outcomes.

CRediT authorship contribution statement

   Lang Shi: Writing – review & editing, Writing – original draft,
   Software, Methodology, Formal analysis, Data curation,
   Conceptualization. Hongchu Zha: Methodology, Formal analysis, Data
   curation. Juan Zhao: Methodology, Formal analysis. Haiqian An:
   Methodology. Hua Huang: Methodology. Yao Xia: Methodology. Ziyu Yan:
   Methodology. Zhixia Song: Methodology. Jiefu Zhu: Writing – review &
   editing, Writing – original draft, Validation, Supervision, Resources,
   Project administration, Investigation, Conceptualization.

Statement of ethics

   All animal care and experimental programs are in the compliance with
   animal management regulations of the Ministry of Health of the People's
   Republic of China and approved by the Ethics Committee of the Three
   Gorges University (202205010T2).

Declaration of competing interest

   The authors have no conflicts of interest to declare.

Acknowledgments