Fluoxetine partially alleviates inflammation in the kidney of socially stressed male C57 BL/6 mice

Stress‐related illnesses are linked to the onset and progression of renal diseases and depressive disorders. To investigate stress‐induced changes in the renal transcriptome associated with the development of depressive behaviors, we generated here a chronic social defeat stress (CSDS) model of C57 BL/6 male mice and then performed RNA sequencing of the kidneys to obtain an inflammation‐related transcriptome. Administration of the antidepressant drug fluoxetine (10 mg·kg−1·day−1) during CSDS induction could partially alleviate renal inflammation and reverse CSDS‐induced depression‐like behaviors. Moreover, fluoxetine also modulated gene expression of stress‐related hormone receptors, including prolactin and melanin‐concentrating hormone. These results suggest that CSDS can induce gene expression changes associated with inflammation in the kidney of C57 BL/6 male mice, and this inflammation can be treated effectively by fluoxetine.

Stress-related illnesses are linked to the onset and progression of renal diseases and depressive disorders. To investigate stress-induced changes in the renal transcriptome associated with the development of depressive behaviors, we generated here a chronic social defeat stress (CSDS) model of C57 BL/6 male mice and then performed RNA sequencing of the kidneys to obtain an inflammation-related transcriptome. Administration of the antidepressant drug fluoxetine (10 mgÁkg À1 Áday À1 ) during CSDS induction could partially alleviate renal inflammation and reverse CSDS-induced depression-like behaviors. Moreover, fluoxetine also modulated gene expression of stress-related hormone receptors, including prolactin and melanin-concentrating hormone. These results suggest that CSDS can induce gene expression changes associated with inflammation in the kidney of C57 BL/6 male mice, and this inflammation can be treated effectively by fluoxetine.
In 2022, the report launched by the World Health Organization (WHO) showed that mental disorders such as depression and anxiety had increased by more than 25% due to the COVID-19 pandemic, adding to the nearly one billion people with a mental disorder [1]. Depression is an already-common and recurrent mental disorder, recognized as one of the leading causes of disability and global disease burden [2]. Depression is a multi-factorial mental disturbance, which is partially attributed to excessive or prolonged psychological stress [3][4][5]. Up to now, the exact mechanisms underlying the pathogenesis of depression remain to be unraveled. However, it has been reported that psychological stress induces a pro-inflammatory response, which might affect peripheral tissues and organs, as well as the brain, and subsequently, inflammation contributes to several psychiatric diseases, such as depression [6,7]. Neurotransmitter serotonin is synthetized from its sole precursor tryptophan and influences a variety of behavioral functions [4,5,8,9]. Inflammation may induce the activation of indoleamine 2,3-dioxygenase, which catalyzes the degradation of tryptophan into kynurenine. Consequently, the level of brain serotonin is reduced, which leads to depression-like symptoms [4,5,8,9]. Hence, drugs that modulate brain serotonin function have been used as antidepressants. Fluoxetine (FLX) is one of the most prescribed selective serotonin reuptake inhibitors for depression, which could alleviate depressive symptoms and provide a neuroprotective effect by reducing neuroinflammation [8][9][10].
Accumulating evidence indicates that months or even years of psychosocial stress, especially lower socioeconomic status and perceived discrimination, are associated with increased risk of acute kidney injury and chronic kidney disease (CKD) progression in human beings across races [11][12][13][14][15][16][17][18][19][20][21][22]. In rodents, immobilization of rats 2 h per day for 5~6 weeks leads to glomerular loss [23] and chronic psychosocial stress induces interstitial nephritis in CBA mice [24]. Such phenomena can be reproduced in NZM2410/J mice which spontaneously develop lupus nephritis [25]. Social defeat stress led to increased deposits of C3 and IgG complexes and macrophage infiltration in the kidney as well as increased serum levels of inflammatory cytokines and anti-dsDNA autoantibodies [25]. Therefore, rodents can be used to analyze the mechanisms underlying the association between chronic psychological stress and the deterioration of kidney function. Indeed, it has been shown that experimental depression induces oxidative stress, altered expression of parathormone receptor and tryptophan hydroxylase, and the rate-limiting enzyme in serotonin synthesis, in the kidney of rats [26][27][28]. In this scenario, research into a genome-wide profile of stress-induced changes in the renal transcriptome that are associated with the development of depressive behaviors is needed. Furthermore, it is of interest to explore the effects of serotonin reuptake inhibitor on stress-induced changes in the kidney.
To answer these questions, we constructed chronic social defeat stress (CSDS) model in C57 BL/6 male mice. Compared with other rodent models of depression, this model better mimics the psychosocial stressors, which are inversely associated with kidney function and effectively reproduces the neuropathological and behavioral phenotypes observed in human depression [29]. We aim to provide a genome-wide view of psychosocial stress-triggered changes in renal transcriptome and clarify the effects of selective serotonin reuptake inhibitor (SSRI) on stress-induced changes in the kidney.

Experimental animals
Adult (retired breeders) male CD-1 mice and 6-week-old male C57BL/6 mice were brought from SPF Biotechnology Co., Ltd (Beijing, China). All mice were maintained under specific pathogen-free conditions with controlled temperature (24 AE 1°C) and humidity (50 AE 10%) and a 12-hlight: 12-h-dark cycle. Mice were acclimatized through 7 days of adaptive feeding. Animal care and experiments were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86-23, revised 1985) and were approved by the ethics committee of the Beijing Institute of Basic Medical Sciences (Permit number: AMMS2020-0356).

CSDS model
CD-1 mice were individually housed and strictly screened for 3 consecutive days. CD-1 mice attacking C57BL/6 mice for at least 2 consecutive days and initiating attack latency ≤30 s were selected for modeling. Male C57BL/6 mice were randomly divided into three groups (n = 6/group): control (Ctrl), CSDS + phosphate-buffered saline (PBS), and CSDS + fluoxetine (FLX). Special cages with a perforated Plexiglas plate separating the space into two halves were used. An intruder C57BL/6 mouse in PBS group or FLX group was directly exposed to a resident CD-1 mouse for 10 min each day. If the CD-1 mouse kept continuous biting even after the C57 BL/6 displayed submissive posture, the defeat bout was immediately terminated. At the end of the frustration, the defeated C57BL/6 mouse was transferred to the other side of the cage so that the two mice could still maintain sensory and olfactory contact [29]. This procedure was repeated for 2 weeks with individual C57BL/6 mouse exposed to different CD-1 mice each day. C57 BL/6 mice in the control group were handled daily and housed in the same type of cages. C57 BL/6 mice in FLX group were intraperitoneally injected once a day with 10 mgÁkg À1 FLX (Cat# 064-04323, Wako Chemical Co. Ltd, Tokyo, Japan) dissolved in PBS; at this dose, this selective serotonin reuptake inhibitor can normalize CSDS phenotypes [30,31], while mice in PBS group injected with the same volume of PBS, during CSDS induction (Fig. 1A).

Forced swim test (FST)
Forced swim test of C57 BL/6 mice was performed as described previously [30][31][32]. Briefly, each C57 BL/6 mouse was put into a cylindrical glass tank with the height of 50 cm and the diameter of 20 cm. The cylindrical tank was cleaned before each mouse was tested. Then, it was filled with tap water which was kept at 23-25°C. The water level is 40 cm above the bottom to ensure that the mouse's tail or feet cannot touch the bottom of the tank. Each mouse was put in the tank for 6 min. The criterion for immobility is that the mouse stops struggling in the water and does nothing but balance its body by the necessary movements to keep its nose out of the water. The accumulated immobility time of mice in the last 4 min was recorded.

Sucrose preference test (SPT)
In this experiment, each mouse was individually housed in its own cage, with free access to food and water. During the first 48 h, the C57 BL/6 mice were habituated to drink from two bottles, one filled with 1% sucrose solution and the other contained tap water. The positions of sucrose solution and the tap water were exchanged every 12 h and counterbalanced across the different cages. On the third day, water and sucrose consumption were measured. The sucrose preference was calculated by the following equation [21]: total liquid intake (g) = sucrose solution intake (g) + tap water intake (g); preference = [sucrose solution intake (g)/total liquid intake (g)] 9 100%.

Histology and immunohistochemistry (IHC)
The mice were anesthetized with pentobarbital sodium and perfused by PBS and 4% paraformaldehyde sequentially. Then, kidneys were removed from mice and fixed in 10% buffered formalin for at least 24 h, dehydrated, and infiltrated with paraffin. Five micrometers paraffin sections were then prepared and stained with hematoxylin and eosin (H & E) for brightfield microscopy. IHC was performed using standard protocols with citrate buffer (pH 6.0) pretreatment. Briefly, formaldehyde-fixed and paraffinembedded kidney sections were incubated with primary antibodies at 4°C overnight and then with horseradish peroxidase-conjugated secondary antibodies at 37°C for 30 min. The sections were finally incubated with diaminobenzidine and counterstained with hematoxylin for detection. The antibody against F4/80 was ordered from BD Bioscience (Cat# 610178, San Jose, CA, USA). Goat antimouse IgG was obtained from Jackson Immunoresearch (Cat# 115-005-003, West Grove, PA, USA).

Bulk RNA sequencing
Total RNA was extracted from kidneys with TRIzol reagent (Cat# 15596026, Life Technologies, Carlsbad, CA, USA). The RNA amount and purity of each sample were quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) with RIN number > 7.0 and confirmed by electrophoresis with denaturing agarose gel. Poly (A) RNA was purified from 1 lg total RNA using Dynabeads Oligo (dT) (Cat# 25-61005, Thermo Fisher Scientific, Waltham, MA, USA) for two rounds. Then, poly (A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (Cat# E6150, New England Biolabs, Herts, UK) at 94°C for 5-7 min. The cleaved RNA fragments were reverse-transcribed to create cDNAs by SuperScript TM II Reverse Transcriptase (Cat#1896649, Invitrogen, Carlsbad, CA, USA), which were next used to synthesize U-labeled second-stranded DNAs with E. coli DNA polymerase I (Cat# M0209, New England Biolabs), RNase H (Cat# M0297, New England Biolabs), and dUTP Solution (Cat# R0133, Thermo Fisher Scientific). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adaptors. Each adaptor contained a The experiment schedule. (B, C) Six-week-old male C57BL/6 mice subjected to CSDS were intraperitoneally injected with FLX (10 mgÁkg À1 ) or PBS of equal volume once a day during chronic stress induction. Then, sucrose preference rate (B) and immobility time in forced swim test (C) were measured. Symbols are values from individual mice (n = 6/group). The results are shown as mean AE standard deviations and were analyzed using PRISM 6.0 (GraphPad) by one-way ANOVA with Dunnett's post hoc analysis. **P < 0.01; ***P < 0.001; ns, not significant. T-base overhang for ligating the adaptor to the A-tailed fragmented DNA. Single-or dual-index adaptors were ligated to the fragments, and size selection was performed with AMPure XP beads (Cat# A63881, Beckman Coulter, Bria, CA, USA). After the heat-labile UDG enzyme (Cat# M0280, New England Biolabs) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95°C for 3 min; 8 cycles of denaturation at 98°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s; and then final extension at 72°C for 5 min. The average insert size for the final cDNA library was 300 AE 50 bp. Finally, we performed the 2 9 150-bp pairedend sequencing (PE150) on an Illumina Novaseq TM 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.

Analysis of bulk RNA-sequencing data
We used FASTP software (v2.2.0) (Haplox, Shenzhen, Guang-Zhou Province, China) to remove the reads that contained adaptor contamination, low-quality bases, and undetermined bases with default parameters. Then, sequence quality was also verified using FASTP [33]. We used HISAT-3N beta to map reads to the GRCm38 mouse reference genome [34]. The mapped reads of each sample were assembled using StringTie with default parameters. Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using the GFFCOMPARE program inside STRING-TIE (the Center for Computational Biology, Johns Hopkins University, MD, USA). After the final transcriptome was generated, StringTie was used to estimate the expression levels of all transcripts by calculating FPKM [total_exon_ fragments/mapped_reads (millions) 9 exon_length (kB)].

Enrichment analysis
For the differentially expressed genes between different groups (|log 2 FoldChange| > 1 and P-value < 0.05), gene set enrichment analysis (GSEA) was conducted to investigate the biological process by the R package CLUSTERPROFILER [35,36]. The Gene Ontology (GO) biological process (BP) gene sets were downloaded from the Molecular Signature database (https://www.gsea-msigdb.org/gsea/msigdb) [37]. P-value < 0.05 was set as the cutoff criterion to screen the prominent biological processes.

Statistical analysis
Quantitative data are shown as mean AE standard deviations and were analyzed using PRISM 6.0 (GraphPad, Boston, MA, USA). One-way ANOVA with Dunnett's post hoc analysis was used to evaluate quantitative variables. P < 0.05 was considered significant.

FLX reverses CSDS-induced depression-like behaviors
Fourteen days of repeated defeats led to anhedonia as reflected by the sucrose preference test (Fig. 1B, P = 0.0043) and psychomotor retardation as reflected by forced swim test (Fig. 1C, P = 0.0008). FLX reversed CSDS-induced anhedonia as reflected by sucrose preference test (Fig. 1B, P = 0.0054). FLX also reversed CSDS-induced psychomotor retardation as reflected by the forced swim test (Fig. 1C, P = 0.0007).

CSDS fails to induce overt renal inflammation
We then analyzed whether CSDS induces macrophage infiltration and IgG deposits in the kidney of male C57 BL/6 mice. Immunohistochemistry revealed no F4/80-positive cells infiltration under CSDS ( Fig. 2A). Kidney tubules were IgG-positive even under steadystate conditions (Fig. 2B). However, CSDS showed no effects on IgG immunohistochemical staining (Fig. 2B). Accordingly, CSDS showed no visible effects on the histology of the kidney (Fig. 2C).

CSDS changes the renal transcriptome
We then employed bulk RNA-sequencing analysis to explore whether CSDS has some detrimental effects on the kidney of C57 BL/6 male and whether all the detrimental effects could be reversed by FLX treatment. The genes were deemed to be differentially expressed if |log 2 FoldChange| > 1 and P-value < 0.05. The data of a mouse in PBS group were too different from others with excessive expression of inflammatory factors and therefore were deleted before further analysis. As expected, CSDS changed the kidney transcriptome with 398 up-regulated genes and 391 down-regulated, among which the top 50 differentially expressed genes are shown (Fig. 3A). To explore the effects of CSDS, GSEA was used to show the biological process of CSDS. The results showed that the CSDS up-regulated genes were involved in processes such as serotonin receptor signaling pathway (P = 0.0135), neuropeptide signaling pathway (P = 0.0318), response to pheromone (P = 0.0181), cytolysis (P = 0.0262), and humoral immune response (P = 0.0220; Fig. 3B). Meanwhile, the down-regulated genes were involved in cytosolic transport (P < 0.0001), muscle cell apoptotic process (P < 0.0001), and cellular component assembly process involved in morphogenesis (P < 0.0001).

FLX treatment changes the renal transcriptome under CSDS
Next, we analyzed how FLX treatment changes the transcriptome in the kidney of male C57 BL/6 mice. Bulk RNA-sequencing analysis revealed that FLX treatment changed the kidney transcriptome under the condition of CSDS with 211 up-regulated genes and 222 downregulated genes, among which the top 50 differentially expressed genes were shown (Fig. 4A). GSEA analysis revealed that FLX treatment down-regulated genes involved in the processes such as RNA modification (P < 0.0001), cytosolic transport (P < 0.0001), hippo signaling (P < 0.0001), and peptidyl-asparagine modification (P = 0.0066). Meanwhile, FLX treatment also up-regulated genes involved in the processes such as mitochondrial transport (P < 0.0001), adaptive thermogenesis (P = 0.0010), thymus development (P = 0.0042), and dicarboxylic acid metabolic process (P < 0.0001; Fig. 4B).

FLX treatment partially reverses the effects of CSDS on renal gene expression
Finally, we compared the transcriptome of the three groups. The results showed that the down-regulated genes of CSDS and the up-regulated ones of FLX treatment shared 94 genes in common (Fig. 5A), while the up-regulated of CSDS and the down-regulated of FLX treatment shared 85 in common (Fig. 5C). The GO_BP enrichment analysis showed that these genes are involved in the processes such as inflammatory response (P = 0.0393, Fig. 5D), immune response (P = 0.0299, Fig. 5B), chemotaxis (P = 0.0102, Fig. 5D), and apoptotic process (P = 0.0001, Fig. 5D). And the inflammation-related genes are shown in

The kidney expresses receptors for stress hormones and serotonin
The molecular basis for the aforementioned changes in the kidney transcriptome was also evaluated. We tried to explore the expression of stress hormone receptors as well as serotonin receptors. Transcripts of glucocorticoid receptor gene Nr3c1 and adrenergic receptor genes Adra1a, Adra1b, Adra1d, Adra2a, Adra2b, Adra2c, Adrb1, Adrb2, and Adrb3 were detected in the kidney of male C57 BL/6 mice, which were not changed by CSDS with or without FLX treatment (Fig. 6A). versus PBS: P = 0.0001) were also detected, which were both up-regulated by CSDS and reversed by FLX treatment (Fig. 6A). On the contrary, transcripts of serotonin receptor genes Htr1b, Htr2a, Htr2b, Htr3a, Htr4, and Htr7 were also detected, which were not changed by CSDS (Fig. 6B). In addition, the expression of parathormone receptor genes (Pth1r and Pth2r) and tryptophan hydroxylase genes (Tph1 and Tph2), was changed insignificantly in the defeated male C57 BL/6 mice (Fig. 6B).

Discussion
The present study provides a genome-wide view of CSDS-triggered changes in renal transcriptome. FLX treatment changes the renal transcriptome under the condition of CSDS, which partially reverses the effects of CSDS on renal gene expression and has additional effects. The molecular basis for these changes might be attributed, at least partially, to the expression of receptors for stress hormones and serotonin in the kidney. CSDS led to depression-like behaviors in male C57 BL/6 mice (Fig. 1B,C) with 398 up-regulated genes and 391 down-regulated in the kidney (Fig. 3A). Recently, it has been revealed that most of the differentially expressed genes in the prefrontal cortex of depressed patients are immediate early genes such as Nr4a3 [38]. In line with these findings, several immediate early genes such as Nr4a3 were up-regulated in the kidney of defeated male C57 BL/6 mice (Fig. 3A). In the brain of male mice after CSDS, it has been demonstrated that the most reproducible transcriptome changes are the expression of hemoglobin genes [38,39], which is believed to provide neuroprotection in response to oxidative stress [40]. Intriguingly, the expression of hemoglobin genes Hba-a1, Hba-a2, Hbbbs, and Hbb-bt was detected in the kidney of male C57 BL/6 mice and showed a trend of up-regulation after CSDS although it failed to reach statistical difference (data not shown). These data are somewhat consistent with the reported oxidative stress in the kidney of rats with experimental depression [26]. In addition, tryptophan hydroxylase protein level was reported to be reduced in the kidney of rats with experimental depression [28]. Accordingly, tryptophan hydroxylase transcripts were detected in the kidney of male C57 BL/6 mice although the effect of CSDS was not significant (Fig. 6B). Furthermore, transcripts of serotonin receptor genes were also detected (Fig. 6B), which can explain the enriched serotonin receptor signaling pathway after CSDS. Besides, the enrichment of CSDS down-regulated genes highlighted cellular transport and cell component assembly process which consume energy (Fig. 3B). Thus, the above changes might reflect the cellular adaptability to cope with the detrimental effects of CSDS on the kidney. However, the cellular adaption seems not enough because the enrichment of CSDS up-regulated genes also highlighted cytolysis (Fig. 3B). Furthermore, the expression of several inflammation-related genes was altered by CSDS (Fig. 5E). Among these genes, S100a8, S100a9, Ccr1, Sphk1, and Cxcl1 have been reported to be involved in inducing renal inflammation [41][42][43][44]. The results show that CSDS leads to an inflammatory transcriptome, even though with no overt effects on the kidney of male C57 BL/6 mice. In line with a previous report [45], the kidney tubules were IgG-positive even under steady-state conditions (Fig. 2B). However, CSDS showed no effects on IgG immunohistochemical staining (Fig. 2B). Accordingly, CSDS showed no visible effects on the histology of the kidney (Fig. 2C).
In the present work, transcripts of glucocorticoid receptor gene, adrenergic receptor genes, prolactin receptor gene, and melanin-concentrating hormone receptor were detected in the kidney of male C57 BL/6 mice (Fig. 6A). Therefore, CSDS might exert detrimental effects on the kidney directly through stress hormones and their receptors. Accordingly, the enrichment of CSDS down-regulated genes highlighted neuropeptide signaling pathway and response to pheromone (Fig. 3B). Furthermore, it has been reported that chronic mild stress leads to increased expression of Prlr and Mchr1 [46,47]. Indeed, CSDS-induced upregulation of Prlr and Mchr1 expression (Fig. 6A). Psychological stress-induced release of prolactin promotes pro-inflammatory immune responses via nuclear factor-jB and interferon regulatory factor-1 [48,49]. On the contrary, melanin-concentrating hormone, also upregulated by psychological stress, has been implicated in the regulation of feeding, emotional processing, and sleep in rodents [50]. And antagonists of melaninconcentrating hormone receptor produce strong antidepressant and antianxiety effects in various models of depression and anxiety [47,50,51]. In addition, it has been reported that CSDS could shift renal circadian clock phase by affecting renal clock gene expression in male C57 BL/6 mice, and the detailed mechanism might be that stress and stress hormones could exert potent effects on peripheral clocks [52]. Indeed, the clock genes Per1 and Cry1 were up-regulated by CSDS (data not shown), which might promote the activation of nuclear factor-jB [53]. Therefore, CSDS exerts detrimental effects on the kidney through various mechanisms.
Fluoxetine treatment reversed CSDS-induced depression-like behaviors (Fig. 1B,C) and partially reversed the effects of CSDS on renal gene expression ( Fig. 5A-E). It has been reported that immediate early genes are the most prominent transcripts modulated by FLX during the induction of depression in different animal models [54]. In line with these observations, the up-regulation of immediate early genes such as Nr4a3 in the kidney of defeated male C57 BL/6 mice was reversed by FLX (Fig. 4A). Recently, a protective role of FLX against oxidative stress in the kidney has been revealed [55]. Accordingly, FLX treatment led to reduced expression of hemoglobin genes in the kidney of defeated male C57 BL/6 mice (Fig. 4A). Furthermore, FLX treatment reversed the aberrant expression of several inflammation-related genes in the kidney of defeated male C57 BL/6 mice (Fig. 5E), which is consistent with the reported anti-inflammatory role of FLX [56,57].
Fluoxetine treatment has been reported to reduce the levels of serum stress hormones such as cortisol and melanin-concentrating hormone [57][58][59], which might alleviate the detrimental effects of CSDS on the kidney. On the contrary, kidney of male C57 BL/6 mice express tryptophan hydroxylase (Fig. 6B), the rate-limiting enzyme in serotonin synthesis [28], so FLX treatment might lead to local serotonin accumulation. Importantly, the kidney of male C57 BL/6 mice express serotonin receptors. Therefore, locally accumulated serotonin can directly regulate gene expression in kidney tissues. Moreover, it has been demonstrated that FLX treatment can reverse chronic mild stressinduced up-regulation of Mchr1 [49]. The present study shows that FLX treatment reversed CSDSinduced up-regulation of both Prlr and Mchr1 (Fig. 6A). In addition, FLX treatment has been revealed to down-regulated clock gene Per1 [60]. Indeed, FLX treatment reversed CSDS-induced upregulation of both Per1 and Cry1 (data not shown). Therefore, FLX treatment can alleviate the detrimental effects of depression through multiple mechanisms.
The present work has some limitations. First, the sample size for behavioral research is small. Since the CSDS model can cause resilience in a small portion of animals [29], it would be better if n ≥ 8. Second, even though the results of the behavior tests FST and SPT verified the facticity of depression, social avoidance, a classical manifestation of depression, and tail suspension test, another indicator of psychomotor retardation, should be assayed. Third, the CSDS model did not take gender effects into consideration. As is known to all, women are prone to be affected by depression than men, because chromosomal and environmental factors contribute to sex differences in vulnerability to depression [61]. Fourth, there is no stress-free group receiving FLX. Although there is shared signature of FLX in stressed and na€ ıve rodents, transcriptomic alterations after FLX treatment in na€ ıve mice or in mice subjected to stress-induced models of depression are quite different [54]. Even though FLX shows a well-established safety profile [62], it might affect kidney potassium channels and perinatal kidney development [63,64]. Without a stress-free group receiving FLX, there is no way to adjudge how FLX might exert nephrotoxicity by affecting gene expression. Finally, ineluctable wounds during the CSDS process should be avoided, which might affect the results of inflammatory response.
Future studies should employ more rodent models of psychological stress and larger sample size to explore whether there are some differences in stress-induced changes in the renal transcriptome between male and female rodents. The establishment of CSDS models in female mice by recent studies [65,66] facilitates achieving these goals. Besides FLX, other antidepressants can be used to explore their potential application to alleviate kidney damage while improving mood. It is important to include a stress-free group receiving the corresponding antidepressant(s). The corresponding findings can incite clinical translational research, for example, a randomized controlled trial about an antidepressant in CKD patients. Furthermore, the present study has identified many differentially expressed genes and enriched pathways, their roles in kidney function and the molecular mechanisms underlying their dynamic expression remain to be explored.

Conclusion
In this work, we propose that chronic psychosocial stress induces inflammatory changes in the kidney, and this could be reversed partially by FLX treatment. The detrimental effects of chronic psychosocial stress might persist and eventually aggravate acute kidney injury or accelerate CKD progression. Antidepressants can be used to alleviate kidney damage while improving mood.