p53/sirtuin 1/NF-κB Signaling Axis in Chronic Inflammation and Maladaptive Kidney Repair After Cisplatin Nephrotoxicity

Chronic inflammation contributes to maladaptive kidney repair, but its regulation is unclear. Here, we report that sirtuin 1 (SIRT1) is downregulated after repeated low-dose cisplatin (RLDC) injury, and this downregulation leads to p65 acetylation and consequent NF-κB activation resulting in a persistent inflammatory response. RLDC induced the down-regulation of SIRT1 and activation of NF-κB, which were accompanied by chronic tubular damage, tubulointerstitial inflammation, and fibrosis in mice. Inhibition of NF-κB suppressed the production of pro-inflammatory cytokines and fibrotic phenotypes in RLDC-treated renal tubular cells. SIRT1 activation by its agonists markedly reduced the acetylation of p65 (a key component of NF-κB), resulting in the attenuation of the inflammatory and fibrotic responses. Conversely, knockdown of SIRT1 exacerbated these cellular changes. At the upstream, p53 was activated after RLDC treatment to repress SIRT1, resulting in p65 acetylation, NF-κB activation and transcription of inflammatory cytokines. In mice, SIRT1 agonists attenuated RLDC-induced chronic inflammation, tissue damage, and renal fibrosis. Together, these results unveil the p53/SIRT1/NF-κB signaling axis in maladaptive kidney repair following RLDC treatment, where p53 represses SIRT1 to increase p65 acetylation for NF-κB activation, leading to chronic renal inflammation.


INTRODUCTION
Nephrotoxicity is the major limiting factor for the clinical use and efficacy of cisplatin, a widely used chemotherapy drug for cancer patients (1)(2)(3)(4). A single high dose of cisplatin induces acute kidney injury (AKI), which involves renal tubular cell injury and death (5)(6)(7). In addition, it induces a robust inflammatory response in kidneys, including the infiltration of immune cells and the production of various pro-inflammatory cytokines (5,8,9). In current clinical settings, cisplatin is often administered in multiple cycles at low doses (10), which lead to chronic kidney problems in a significant portion of cancer patients (11). In animals, repeated low dose cisplatin (RLDC) treatment leads to chronic renal pathologies characterized by tubular degeneration, renal inflammation, and interstitial fibrosis, which are associated with a progressive decline of renal function (12)(13)(14)(15)(16)(17). However, the mechanism of RLDCinduced chronic renal pathologies remains unclear.
Following AKI, some tubular cells are injured and die, but the surviving cells have the capacity to regenerate and repair the damaged renal tubules. Complete repair can restore the integrity and function of renal tubules for a full recovery. However, after severe AKI or repeated episodes of injury, kidney repair is often incomplete or maladaptive, resulting in atrophic tubules that produce various factors to stimulate inflammation, vascular dropout, myofibroblast expansion and matrix deposition for renal fibrosis (18)(19)(20)(21). RLDC is expected to induce maladaptive kidney repair for its repeated toxicity to renal tubular cells. Indeed, following RLDC treatment, mice showed tubular degeneration, chronic inflammation, and varied degrees of interstitial fibrosis depending on the models (12)(13)(14)(15)(16). Remarkably, the pathological changes were associated with a progressive decline of renal function (15,16).
It is generally understood that renal inflammation persists in, and contributes to, maladaptive kidney repair; but the mechanism of chronic renal inflammation during maladaptive kidney repair remains elusive (18)(19)(20)(21)(22). Nuclear factor-kB (NF-kB) is a family of "master" transcription factors that are responsible for immune and inflammatory gene expression (23). This family consists of five structurally related members, including NF-kB1 (also called p50), NF-kB2 (also called p52), RelA (also called p65), RelB, and c-Rel, which form hetero-or homo-dimers, i.e. NF-kB. In unstimulated cells, NF-kB is sequestered in the cytoplasm by its inhibitory proteins IkBs (24). Upon stimulation, IkBs is phosphorylated by the IkB kinase (IKK) with consequent ubiquitination and proteasomal degradation (23). Following IkB degradation, NF-kB is released and translocated to the nucleus to induce target gene transcription (25). In the nucleus, p65 (a critical component of the NF-kB complex) is acetylated and the acetylation status of specific lysine residues in p65 affects both the DNA-binding ability and transcriptional activity of NF-kB for various proinflammatory cytokines, such as Tnf-a, Il-8, Il-6, and Il-1b (24,26). NF-kB plays a critical role in renal inflammation in kidney diseases (27). However, the role and regulation of NF-kB in maladaptive kidney repair remains largely unclear.
Mammalian sirtuin 1 (SIRT1) is a protein deacetylase of the highly conserved nicotinamide adenine dinucleotide (NAD + )dependent sirtuin family that is ubiquitously expressed in mammalian cells (28). In addition to its bona fide function of histone deacetylation, SIRT1 also deacetylates lysine residues of various proteins to participate in the aging process (29), inflammation (30), cancer (31), metabolic (32) and neurodegenerative diseases (33). In the kidney, SIRT1 and other sirtuins play important roles in renal function and physiology, and their dysregulation contribute to the pathogenesis of kidney diseases (34). For example, activation of SIRT1 reduced the acetylation of Smad3 and ameliorated fibrosis in 5/6 nephrectomy rats probably by suppressing TGF-b/Smad3 signaling (35). In unilateral ureteral obstruction (UUO), SIRT1 was shown to be an important protective factor for renal medullary interstitial cells against oxidative stress partly by regulating of COX2 (36). In diabetic models, SIRT1 prevent renal fibrosis by suppressing HIF1a, GLUT1, and SNAIL (37). However, the role and regulation of SIRT1 in maladaptive kidney repair remain unclear.
In the present study, we demonstrate that SIRT1 is repressed by p53 during maladaptive kidney repair following cisplatin nephrotoxicity, and the down-regulation of SIRT1 contributes to p65 acetylation and consequent NF-kB activation, resulting in renal inflammation and fibrosis. Therapeutically, targeting the p53/SIRT1/NF-kB signaling axis may improve kidney repair and recovery.

Animal Models
Male C57BL/6 mice (8 weeks) were purchased from SJA Laboratory Animal Corporation (Hunan, China) and maintained in the animal facility of the Second Xiangya Hospital. Mice received saline vehicle or 8 mg/kg cisplatin (Hansoh Pharma, Jiangsu, China) intraperitoneally once a week for 4 weeks (15). For intervention, SRT1720 (a small molecule activator of SIRT1 purchased from APExBIO, Houston, USA) was administered to mice at 100 mg/kg/ day (38) by intraperitoneal injection for 7 days after the last cisplatin dose. To inhibit p53, pifithrin-a (APExBIO, Houston, USA) was administered to mice at 2.2 mg/kg/day by intraperitoneal injection for 7 days after the last cisplatin dose. Animals were sacrificed at 4 and 8 weeks after the last cisplatin dose. All animal experiments were performed according to a protocol approved by the Institutional Committee for the Care and Use of Laboratory Animals of the Second Xiangya Hospital at Central South University.

Cell Culture and Treatments
The Boston University mouse proximal tubular cell line (BUMPT) was originally obtained from Dr. Lieberthal (Boston University) and cultured as previously (15). The cells were subjected to four cycles of treatment with 0, 0.5, 1, or 2 mM cisplatin (15). Each cycle consisted of 7 h of cisplatin incubation and 17 h of recovery in cisplatin-free medium. For intervention, BUMPT cells were initially subjected to 4 cycles of cisplatin treatment. After the final dose, cells were incubated with 100mM TPCA1, 20mM RSV, 2.5mM SRT1720, or 25mM pifithrin-a (all purchased from APExBIO, Houston, USA) for 17h in cisplatinfree medium. Small interference RNA (siRNA) specific against the mouse SIRT1 gene, p53 gene and scrambled control siRNA were synthesized by RiboBio (Guangzhou, China). Cells were transfected with 50 nM Sirt1 siRNA, Trp53 siRNA or control siRNA for 24h using riboFECT TM CP Transfection Kit (RiboBio, Guangzhou, China).
Transcutaneous Measurement of Glomerular Filtration Rate (GFR) mice were anesthetized with isoflurane using an anesthesia machine, which contains the breathing circuit, air intake pump, recovery device and induction box. Then, a transdermal GFR monitor (MediBeacon, Mannheim, Germany) was attached to hair-shaved skin of anesthetized mice. FITC-sinistrin (7mg/ 100g b.w.) was injected through the tail vein. Data analysis is based on the elimination kinetic curve of FITC-sinistrin.

Masson Trichrome Staining
Kidney sections were stained using the kit from Servicebio (Wuhan, China). For quantification, 10 positive collagenstained fields (100 magnification) were randomly selected from each section and analyzed by Image-Pro Plus 6.0 to assess the percentage of fibrotic area.

Immunofluorescence
Immunofluorescence staining was conducted as previously (39). After fixation with cold methanol: acetone (1:1), the cells were incubated with a blocking solution containing 10% goat serum, followed by overnight incubation with primary antibodies, and finally with secondary antibodies at 37°C for 1 hour. Primary antibodies for immunofluorescence included those for p65 (8242, Cell Signaling Technology), Collagen I (AF7001, Affinity), p-p65 (3033, Cell Signaling Technology), SIRT1 (8469, Cell Signaling Technology). Cy3-labeled goat anti-rabbit IgG and FITC-labeled goat anti-rabbit IgG were used as secondary antibodies. The cells were counter-stained with Hoechst to show the nucleus and examined on a Zeiss LSM780 confocal microscope (Oberkochen, Germany).

Extraction of Nuclear and Cytoplasmic Proteins
The extraction was done with a kit was purchased from Beyotime (Shanghai, China). Briefly, cells were centrifuged to collect the cell pellet, which was mixed with PMSF-containing cytoplasmic protein extraction reagent A, vortexed vigorously and then incubated in ice bath. Cytoplasmic protein extraction reagent B was then added. After vortexing, the mixture was centrifuged at 12000g for 5 minutes at 4°C to collect the supernatant as the cytoplasmic fraction. Then, the pellet was re-suspended in the nucleoprotein extraction reagent by vigorous vortexing (15-30s every 1-2 minutes for a total of 30 minutes), and then centrifuged at 12000g for 10 minutes at 4°C to collect the supernatant as the nuclear proteins.

Quantitative Real-Time PCR
Total RNA was extracted with TRIzol reagents (CWBIO, Jiangsu, China) according to the manufacturer's protocol. cDNA was synthesized using the PrimeScript RT reagent Kit (TaKaRa, Japan). Quantitative real-time PCR was performed using the TB Green Premix Ex Taq II reagent (TaKaRa, Japan) on a LightCycler96 Real-Time PCR System. Relative expression was normalized to the expression levels of GAPDH. The primer sequences for PCR are listed in Table 1.

ChIP Assay
ChIP assay was performed with the ChIP-IT ® High Sensitivity kit (Active Motif, USA) according to the manufacturer's instruction. Briefly, cells were cross-linked with 1% formaldehyde followed by incubation in glycine stop-fix solution. Then, the cells were lysed with the lysis buffer containing protease inhibitor cocktail and deacetylase inhibitors. Purified chromatin was sonicated and incubated with an indicated immunoprecipitation antibody anti-p53 antibody (Cell Signalling Technology, 2524). The resultant immunoprecipitate was subjected to qPCR amplification of putative p53 binding sequences using specifically designed primers. The value of qPCR was normalized with input DNA for comparison. The following primers were used for p53 binding site detection: forward GCGCCAGAGGCCGCTGA and reverse CGGCTGCGGGAGATTTAAACC.

Statistics
Statistical analysis was performed using the GraphPad Prism software. Multiple group comparison was analyzed with ANOVA followed by Tukey's posttests. Statistical differences between two groups were determined by two-tailed unpaired or paired Student's t-test. The value of P < 0.05 was considered significantly different.

RLDC Treatment Induces Chronic Kidney Injury and Activates NF-kB During Maladaptive Kidney Repair
One month after RLDC treatment, mice had a significant decline of renal function as measured by the clearance rate of FITClabeled sinistrin and the calculated GFR ( Figures 1A, B). These mice showed clear signs of renal tubular damage, including renal tubule expansion, slight loss of brush border, cast formation and increased inflammatory cell infiltration ( Figure 1C). Tubular damage was further semi-quantified in cortex and outer medulla ( Figure 1D). Along with tubular injury was a chronic inflammatory response. The expression of multiple proinflammatory cytokines, including interleukins (Il-1b, -6, -8) and tumor necrosis factor (TNF)-a, was markedly increased in post-RLDC kidney tissues (Supplemental Figure 1A). Moreover, there was a dramatic increase of macrophages shown by F4/80 staining (Supplemental Figures 1B, C). These results indicate that the inflammatory response in injured tubule cells may contribute to chronic renal inflammation and maladaptive repair following RLDC treatment.
NF-kB is a classical transcription factor for the expression of inflammatory cytokines. Upon activation, p65, a key component of NF-kB, is phosphorylated and translocated into the nucleus for downstream gene expression. We detected p65 phosphorylation in post-RLDC kidneys, which was accompanied by increases of fibrosis markers like fibronectin (FN) ( Figures 1E, F). In immunofluorescence, a considerable portion of p65 moved to the nucleus in renal tubule cells after RLDC treatment ( Figure 1G). For in vitro experiments, we recently established a cell model of RLDC treatment in BUMPT cells (15). Consistently, RLDC induced p65 phosphorylation as well as FN and COL-1 in BUMPT cells in a dose-dependent manner ( Figure 1H). Moreover, immunofluorescence detected nuclear accumulation of p65 in post-RLDC cells ( Figure 1I). Immunoblot analysis of nuclear and cytosolic fractions demonstrated a notable increase of p65 in cell nucleus, which was accompanied by a slight increase of p65 in the cytosol ( Figure 1J). Also in this cell model, RLDC also induced the expression of Il-1b, Il-6, Il-8, and Tnf-a ( Figure 1K), suggesting that injured tubule cells might be a major resource of pro-inflammatory cytokines after RLDC treatment. These results indicate that RLDC promotes p65 expression and nuclear accumulation for NF-kB activation, accompanied by the induction of a pro-fibrotic phenotype in renal tubular cells.

TPCA-1 Reduces the Expression of Pro-Inflammatory Cytokines and Fibrotic Phenotypes in RLDC-Treated Renal Tubular Cells
To determine the role of NF-kB, we evaluated the effect of TPCA-1, which suppresses NF-kB by selectively inhibiting IKK-2 (41,42). As shown in Figures 2A, B, TPCA-1 markedly ameliorated the fibrotic changes in RLDC-treated tubular cells, as indicated by reduced expressions of FN and VIM. Immunofluorescence further verified the inhibitory effect of TPCA-1 on COL-1 expression ( Figures 2C, D). In addition, TPCA1 attenuated the expression of multiple pro-inflammatory cytokines, including Il-1b, Il-6, Il-8, and Tnf-a ( Figure 2E). These data suggest that RLDC triggers aberrant activation of NF-kB in renal tubular cells to stimulate a robust inflammatory response and pro-fibrotic changes.

RLDC Induces Down-Regulation of SIRT1 and Consequent Acetylation of p65
We then investigated the mechanism of NF-kB activation induced by RLDC. SIRT1 is a powerful deacetylase that has been reported to deacetylate p65 on lysine-310 to inhibit NF-kB (43). Recent work has implicated SIRT1 as a protective factor in cisplatininduced acute kidney injury (44). We therefore hypothesized that SIRT1 may regulate NF-kB by deacetylating p65 during the development of chronic kidney problems after RLDC treatment. In immunohistochemical staining, SIRT1 was mainly present in the nucleus of tubular cells ( Figure 3A) and, after RLDC treatment, the SIRT1 signal was decreased to half of control ( Figure 3B). Importantly, decreased expression of SIRT1 was accompanied by increased Ac-p65 level ( Figures 3C, D). This expression pattern was further confirmed in BUMPT cells where RLDC markedly decreased SIRT1 expression in a cisplatin dosedependent manner, while increasing Ac-p65 ( Figures 3E, F). These results suggest that RLDC may decrease SIRT1 to promote p65 acetylation, contributing to NF-kB activation in post-RLDC kidneys and renal tubular cells.

SIRT1 Activator Reduces p65/NF-kB Activation and Ameliorates Kidney Inflammation and Fibrosis After Post-RLDC Treatment
Next, we evaluated the therapeutic potentials of SIRT1 agonists in RLDC-treated mice. To this end, after RLDC treatment, the

Gene
Forward Reverse SIRT1 agonist SRT1720 (S) was injected daily into the mice for one week. One group of animals were sacrificed immediately after S treatment to examine its effects on kidney injury, SIRT1 and p65 acetylation, while another group of animals were terminated 3 weeks later to examine the long-term effect of SRT1720 on chronic renal inflammation and interstitial fibrosis (Supplemental Figure 2A).
In both immunoblot and immunostaining analyses, RLDC induced a marked down-regulation of SIRT1 in kidneys, which was partially but significant prevented by SRT1720. Notably, SRT1720 inhibited p65 activation, manifested by the decrease of Ac-p65 and nuclear p65 (Figures 4A-D). Three weeks later, the mice injected with SRT1720 showed reduced inflammatory cytokine production ( Figure 4E  and ameliorated renal interstitial fibrosis ( Figures 4F-I). In addition, SRT1720 reduced kidney atrophy, tubular damage, and renal function decline in post-RLDC kidneys (Supplemental Figures 2B-F). Collectively, these findings suggest that activation of SIRT1 may suppress the proinflammatory and pro-fibrotic changes in post-RLDC kidneys to improve kidney repair.
To determine how the SIRT1/NF-kB signaling axis affects tubular repair in post-RLDC kidneys, we examined tubular cell proliferation and senescence. We first examined the expression of Ki67, a marker of cell proliferation in renal tubules. A representative image of Ki67 staining and the quantification are shown in Supplementary Figures 4A, B. As expected, the control kidney had very few Ki67 positive cells. After RLDC, there was a 2fold increase in renal tubular Ki67 staining, indicating renal tubular cell proliferation for kidney repair. It is worth noting that SRT1720 (SIRT1 agonist) increased Ki67 staining in renal tubules, indicating that renal repair was enhanced. Premature senescence plays an important role in maladaptive kidney repair including renal fibrosis. In order to explore the influence of SIRT1 activation on premature senescence, we performed SA-b-Gal staining, which is a standard marker of senescence. As shown in Supplementary Figures 4C, D, the renal tubules after RLDC treatment showed a significant increase of SA-b-Gal staining, mainly in the renal cortex, which was markedly reduced by SRT1720, suggesting the anti-senescence function of SIRT1 in this model. Furthermore, Ki67-cells with high g-H2AX lesion density (four or more lesions per nucleus) indicate renal tubular senescence (45). SRT1720 reduced g-H2AX + /Ki67cells in the kidney (Supplementary Figures 4E, F), and reduced the expression of g-H2AX in immunoblot analysis ( Supplementary  Figures 4G, H). Together, these new results indicate that SIRT1 activation by SRT1720 enhances renal tubular proliferation and ameliorates renal senescence after RLDC, providing further mechanistic insights into the regulation of maladaptive kidney repair by the SIRT1/NF-kB axis.

SIRT1 Ameliorates Fibrotic and Inflammatory Phenotypes in RLDC-Treated Cells
In vitro, resveratrol (R) and SRT1720 (S) could significantly increase SIRT1 expression in both RLDC-treated and control BUMPT cells ( Figure 5A). Importantly, these SIRT1 activators reduced the expression of Ac-p65 and p-p65 during RLDC treatment, and reduced nuclear accumulation of p65 as shown by immunofluorescence staining (Figures 5A-C). Consistently, immunoblot analysis of cellular fractionations showed that SIRT1 activators decreased nuclear p65 whereas increased cytosolic p65 in RLDC-treated cells ( Figures 5D, E), which indicates that activation of SIRT1 leads to p65 deacetylation resulting in the prevention of p65/NF-kB activation during RLDC treatment. To examine the effects of SIRT1 activators on RLDC-induced maladaptive repair responses, persistent fibrotic changes were induced in BUMPT cells, featured by increased FN and VIM expression. These fibrotic changes were markedly attenuated by SIRT1 activators (Figures 5F, G). Moreover, SIRT1 activators reduced the expressions of pro-inflammatory cytokines in RLDC-treated cells, especially Il-8 and Tnf-a ( Figure 5H). Thus, these findings support that activation of SIRT1 ameliorates the pro-inflammatory and profibrotic changes in RLDC-treated cells.
We further examined the effects of SIRT1 knockdown. Transfection of SIRT1 siRNAs (si1 and si2) attenuated SIRT1 expression in BUMPT cells. Notably, these cells had 3-4 fold higher expression of Ac-p65 ( Figures 6A, B), and showed more nuclear accumulation of p65 ( Figure 6C). Knockdown of SIRT1 aggravated the expression of fibrotic markers (eg. FN and VIM) ( Figures 6D, E) and pro-inflammatory cytokines (eg. Il-1b, Il-6, Il-8, Tnf-a) ( Figure 6F) in RLDC-treated cells. These results further support the conclusion that down-regulation of SIRT1 during maladaptive kidney repair contributes to NF-kB activation, renal inflammation, and fibrosis.
p53 Mediates the Down-Regulation of SIRT1 After RLDC Treatment We focused on p53 to understand the mechanism of SIRT1 down-regulation in post-RLDC kidneys mainly based on two facts: 1) RLDC induced a significant decrease in SIRT1 mRNA in BUMPT cells indicating SIRT1 down-regulation at the transcription level ( Figure 7E), and p53 has been reported to regulate SIRT1 transcription (46); and 2) p53 is known to mediate cisplatin-induced AKI (47,48), and we detected p53 activation in post-RLDC mouse kidneys ( Figure 8E). We examined the effect of pifithrin-a (PF), a commonly used pharmacologic inhibitor of p53. As shown in Figures 7A, B, PF reduced the increase of p53 and p-p53 in RLDC-treated cells. Importantly, PF restored SIRT1 expression in these cells at both protein ( Figures 7C, D) and mRNA ( Figure 7E) levels. Bioinformatics analysis using JASPAR database (http://jaspar. genereg.net/) identified a potential p53 binding site ( Figure 7F) on the promoter of Sirt1 gene. ChIP analysis showed that RLDC specifically increased the binding of p53 to the Sirtl gene promoter at this site ( Figure 7G). As for the SIRT1/NF-kB pathway, inhibition of p53 with PF also reduced p65 phosphorylation and acetylation ( Figures 7H, I). For further validation, we also knocked down p53 in mouse renal tubular cells (Supplementary Figure 5A) and found that the results were consistent with pharmacological inhibitors, i.e., p53 knockdown alleviated RLDC-induced SIRT1 reduction and p65 activation ( Supplementary Figures 5B-E).
In mice, PF also inhibited p53 in post-RLDC kidneys and reduced SIRT1 down-regulation and p65/NF-kB activation including phosphorylation and acetylation ( Figure 8). Collectively, these results indicate that p53 mediates SIRT1 down-regulation during post-RLDC kidney repair by directly repressing Sirt1 gene transcription.

DISCUSSION
RLDC regimen is a common way to reduce the risk of nephrotoxicity in cisplatin chemotherapy, but, even with extreme clinical cautions, the incidence of acute and chronic kidney problems remains significant. Recent studies have verified the chronic effects of RLDC on kidneys in animal models; however, the underlying mechanism is largely unclear. There are three major findings in our current study as we have shown in Figure 8J: 1. RLDC induces aberrant NF-kB activation in renal tubular cells that contributes to chronic kidney injury by triggering the production of pro-inflammatory cytokines for a persistent inflammatory response; 2. SIRT1 is down-regulated in post-RLDC kidneys and this downregulation contributes to NF-kB activation and associated chronic represses NF-kB signaling by deacetylating its key component p65. Following RLDC treatment, p53 is activated to repress SIRT1, leading to p65/NF-kB acetylation and activation inducing the transcription of various pro-inflammatory cytokines for renal inflammation and fibrosis. By inhibiting p53, pifithrin-a may prevent SIRT1 downregulation and NF-kB activation. As SIRT1 agonists, SRT1720 and resveratrol activate SIRT1, resulting in p65 deacetylation to suppress NF-kB and renal inflammation and fibrosis. Data are expressed as mean ± SEM. *P<0.05 vs. the control group (Con), #P < 0.05 vs. RLDC with vehicle solution group (RLDC + ve).
inflammation and kidney injury; 3. p53 mediates the downregulation of SIRT1 by repressing its gene transcription in post-RLDC kidneys. Together, these results unveil the p53/SIRT1/NF-kB signaling axis in chronic renal inflammation and pathology during maladaptive kidney repair, highlighting potential therapeutic targets. NF-kB is a transcription factor that plays important roles in the regulation of inflammation, cell death, survival, proliferation and differentiation (49,50). In kidneys, NF-kB promotes inflammation by inducing the expression of pro-inflammatory cytokines in multiple renal disease settings (27). Inhibition of NF-kB in acute nephrotoxicity ameliorates kidney injury and tubular necrosis by attenuating inflammation (51), but the role of NF-kB in maladaptive kidney repair after AKI is less unclear. In this study, we demonstrate for the first time the continuous activation of NF-kB after RLDC treatment, accompanied by chronic inflammation, renal tubular damage and atrophy, and interstitial fibrosis. Inhibition of NF-kB resulted in a decrease in the expression of pro-inflammatory factors in tubule cells, less tubular damage and less fibrosis, supporting a pivotal role of NF-kB in maladaptive kidney repair following RLDC treatment.
Emerging studies have suggested a protective role of SIRT1 in the pathogenesis of renal fibrosis. In UUO, SIRT1 expression or activation in renal medullary interstitial cells attenuated the expression of COX2 and the subsequent renal fibrogenesis, linking SIRT1 in renal interstitial cells to oxidative stress signaling and renal fibrosis (36). In addition, SIRT1 activation could inhibit the TGFb/Smad3 pathway in 5/6 nephrectomy and obstructed kidneys through epigenetic regulation of Smad3, leading to the amelioration of kidney fibrosis (35,52). Nevertheless, it is known that, following injury, renal tubular cells secrete pro-inflammatory and pro-fibrotic cytokines for maladaptive kidney repair including fibrosis. Accordingly, SIRT1 may ameliorate these responses to improve kidney repair. Our current study provides insights into this mechanism. We show that SIRT1 can deacetylate p65 (a critical component of NF-kB) to prevent NF-kB activation in renal tubular cells. During kidney repair after RLDC treatment, SIRT1 was dramatically down-regulated, resulting in increased p65 acetylation and NF-kB activation leading to the production of various pro-inflammatory cytokines for maladaptive kidney repair. It is worth noting that SIRT1 may deacetylate other target proteins as well. For example, SIRT1 may alleviate cisplatininduced renal senescence by deacetylating p53 (53). Future research needs to delineate the possible crosstalk between NF-kB signaling and other pathways in terms of SIRT1 regulation during maladaptive kidney repair.
To understand the mechanism of SIRT1 down-regulation following RLDC treatment, we focused on p53, which was implicated in transcriptional regulation of SIRT1 previously (46). In our study, RLDC induced p53 activation and the decrease of SIRT1 transcription in BUMPT cells and mouse kidneys (Figures 7, 8). Moreover, inhibition of p53 with pifithrin-a prevented SIRT1 down-regulation (Figures 7, 8).
ChIP analysis further showed that RLDC specifically increased the binding of p53 to the promoter of the Sirt1 gene ( Figure 7G), indicating a direct regulation of SIRT1 by p53. Pifithrin-a also reduced the phosphorylation and acetylation of p65 after RLDC treatment ( Figures 7H and 8H). All these results support a role of p53 in SIRT1 down-regulation during maladaptive kidney repair after RLDC treatment. Interestingly, SIRT1 may directly deacetylate p53 (53), suggesting a feedback mechanism to control SIRT1 expression.
The current study also suggests therapeutic potentials by targeting the p53/SIRT1/NF-kB signaling axis. First, considering the role of inflammation in the maladaptive repair process, inhibiting inflammation is a logical strategy for improving kidney repair. In this regard, Ravichandran et al. showed that depletion of CD4 T cells did not protect against lowdose cisplatin-induced AKI or subsequent renal fibrosis in mice with cancer, but it led to a weakened anti-cancer effect of cisplatin (17). In clinical trials, monoclonal antibodies that neutralize inflammatory factors have been used to treat kidney diseases. For example, CCR2 antagonists were used in the treatment of type 2 diabetes in a phase I clinical trial (54,55). However, maladaptive kidney repair likely involves multiple inflammatory factors, and the key pathogenic inflammatory factors remain to be identified by experimental and clinical studies. In this regard, targeting of NF-kB, the "master" transcription factor for a myriad of pro-inflammatory cytokines, would shut down overall inflammation to prevent maladaptive kidney repair and facilitate normal kidney repair or functional recovery. Notably, the current study has unveiled SIRT1 as a negative regulator of NF-kB and pharmacological activation of SIRT1 resulted in deacetylation of p65 and suppression of NF-kB, which were associated with reduced renal inflammation and improved kidney repair. These observations support the therapeutic potential by activating SIRT1 and/or inhibiting NF-kB.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Ethics Committee of the Second Xiangya Hospital of Central South University.

AUTHOR CONTRIBUTIONS
ZD and YF designed this study; YF performed most of the experiments; YF, YW, YL, and CT contributed to data analysis; ZD and YF analyzed results; GC and JC provided suggestions for manuscript preparation; ZD and YF contributed to manuscript writing. All authors contributed to the article and approved the submitted version.