Global proteome and phosphoproteome characterization of sepsis-induced kidney injury.

Sepsis-induced acute kidney injury (S-AKI) is the most common complication in hospitalized and critically ill patients, highlighted by a rapid decline of kidney function occurring a few hours or days after sepsis onset. Systemic inflammation elicited by microbial infections is believed to lead to kidney damage under immunocompromised conditions. However, while AKI has been recognized as a disease with long-term sequelae, partly due to the associated higher risk of chronic kidney disease (CKD), the understanding of kidney pathophysiology at the molecular level and the global view of dynamic regulations in situ after S-AKI, including the transition to CKD, remains limited. Existing studies of S-AKI mainly focus on deriving sepsis biomarkers from body fluids. In the present study, we constructed a mid-severity septic murine model using cecal ligation and puncture (CLP), and examined the temporal changes to the kidney proteome and phosphoproteome at day 2 and day 7 after CLP surgery, corresponding to S-AKI and the transition to CKD, respectively, by employing an ultrafast and economical filter-based sample processing method combined with the label-free quantitation approach. Collectively, we identified 2,119 proteins and 2,950 phosphosites through multi-proteomics analyses. Among them, we identified an array of highly promising candidate marker proteins indicative of disease onset and progression accompanied by immunoblot validations, and further denoted the pathways that are specifically responsive to S-AKI and its transition to CKD, which include regulation of cell metabolism regulation, oxidative stress, and energy consumption in the diseased kidneys. Our data can serve as an enriched resource for the identification of mechanisms and biomarkers for sepsis-induced kidney diseases.


Introduction
Acute kidney injury (AKI) is characterized by a rapid decline of renal function resulting in elevated serum creatinine levels with or without decreased urine output 1 . It is estimated that the downloaded from UniProt Knowledgebase (https://www.uniprot.org/) was used for the database search. For global proteome analysis, the following parameters were applied: 10 ppm and 20 ppm mass tolerances for precursor and fragments, respectively; trypsin as enzyme with two missed cleavage sites; protein N-terminal acetylation and methionine oxidation as variable modifications; cysteine carbamidomethylation as a fixed modification; peptide length with at least 7 amino acids.
False discovery rate (FDR) was set at 1% for both proteins and peptides. For global proteome quantitation, the MaxQuant output result (proteinGroups.txt) was first filtered to exclude those 'Only identified by site, 'Potential contaminant', and 'Reversed' hits, and then log2-transformed.
Downstream data analyses such as missing value imputation, Hierarchical clustering, Principal Component Analysis (PCA), t-tests, correlation, and volcano plots were performed in the Perseus environment using default parameters (version 1. 6 the Cytoscape environment (version 3.8.0) 24 . The interaction score was set to 0.9, the highest confidence cutoff, to retrieve potential interactions. The 'Load Enrichment Data' option was enabled during this process to retrieve functional enrichment (minimum significance threshold

Results
Characterization of kidney injury after S-AKI. Given that S-AKI is the most common complication associated with sepsis in the clinic, we aimed to explore its pathogenesis in detail.
We created a moderate S-AKI mouse model in which the mice would survive to progress to chronic kidney disease. CLP surgery was performed by placing a 2-0 silk ligature at 1 cm from the cecal tip as described previously (Figure 1A), with mice sacrificed at day 2 or day 7 12 . Only 1 out of 20 mice that went through the procedure died before the entire experiments were finished, indicating we did not induce severe sepsis. Next, we examined if the CLP procedure successfully induced AKI using rapid elevation of serum creatinine (SCr) as an established biomarker of AKI 25 . We observed that SCr was significantly increased to an average of 0.33 mg/dl at 2 days after by guest on September 23, 2020 the CLP procedure, a nearly 3-fold increase compared to the sham control group. By 7 days after CLP, SCr returned to a relatively low level ( Figure 1B). We also investigated the expression of neutrophil gelatinase-associated lipocalin protein (NGAL; also known as lipocalin 2, Lcn2), a validated predictor for kidney damage. Under ischemic, septic, or post-transplant AKI, NGAL's expression in kidney tubules is often rapidly increased 26 . NGAL expression increased dramatically in renal tissues at day 2 after CLP compared to the sham control, with a reduction by day 7 (Figure 1C and 1D), indicating that CLP caused a definite injury to the kidneys.
Consistent with this, periodic acid-Schiff (PAS) staining illustrated cell vacuolation, necrosis, and cellular debris present near blood vessels in the diseased kidneys at day 2 after CLP ( Figure 1E), which are consistent with previous observations of septic kidneys 27 . Little to no differences in histological changes were observed between day 2 and day 7 after CLP. In contrast to S-AKI, ischemia-reperfusion associated AKI usually causes more profound histological changes, as reported by our previous study 28 .
It has been known that cell apoptosis and inflammatory cell infiltration are two major cellular processes in the development and progression of AKI 29 . To investigate this, we examined two cell apoptosis-related proteins, Bcl2-associated agonist of cell death protein (Bad) and FASassociated death domain protein (Fadd) 29,30 . Bad appeared to remain unchanged (p > 0.05) across the three time points, whereas Fadd showed mild increase at day 7 in comparison to sham control ( Figure 1F through 1H). Consistently, the expression of tumor necrosis factor-α (TNF-α) displayed a similar trend to Fadd in the S-AKI kidneys by western blot ( Figure 1I) and enzymelinked immunosorbent assay (ELISA) ( Figure 1J). As areas of necrosis were observed in our histological analysis (Figure 1E), we tested the effector proteins of pyroptosis 31 , whose activity has recently been associated with acute kidney injury 32 . Gasdermin D, the effector molecule that forms the membrane pore and mediates pyroptosis 31 , was significantly increased in the injured kidneys at day 7 after CLP ( Figure 1, K and L). Cleaved caspase-1, which mediates Gsdmd by guest on September 23, 2020 cleavage and activation, was also significantly increased, along with phosphorylated ASC (apoptosis-associated speck-like protein containing a CARD; also known as Pycard), which regulates caspase-1 activation (Figure 1, M and N). In addition, phosphorylation of ERK, whose activation mediated LPS-induced inflammasome formation 33 was significantly increased as well (Figure 1, M and N). Collectively, our data strongly suggest canonical pyroptosis activation and inflammasome formation, and that a delayed biological response to septic injury in kidney dominates the transition from AKI to CKD.
The global proteomic view of the septic kidneys. To gain an unbiased understanding of the underlying molecular determinants that modulate S-AKI, we analyzed the global profile of the renal tissue proteomes of mice at day 0, 2 and 7 post-CLP following a label-free quantitative approach (Figure 2A). The global kidney proteome was analyzed using a 220-min single-run LC-MS/MS approach to balance the throughput and proteomic depth. Collectively, 2,119 proteins were quantified with a less than 1% false discovery rate (FDR), and nearly 90% of them were detected across all three groups (Supplementary Table S1). The correlation between biological replicates (i.e. different mice) within the same group was high (R 2 = 0.95 ± 0.02, n = 45) ( Figure   2B), which validated the reliability of our CLP model and the reproducibility of the sample processing approach. We also noticed that the correlation between different groups was relatively high as well (R 2 = 0.93 ± 0.02, n = 108). These data indicate that the CLP procedure induced lowto mid-grade sepsis, likely only allowing a minimal amount of feces to extrude from the perforation site. Interestingly, the overall unfiltered proteomics data enabled the unsupervised classification of the kidneys according to their global profiles, which clustered day-2 mice tightly together while control and day-7 samples were intermixed (Supplemental Figure S1). The data suggested that the kidney global proteome has undergone systematic changes after moderate septic injury, and may have reshaped back closer to healthy kidneys a week after CLP.
To define the proteins that were differentially regulated in response to septic injury, and determine underlying modulatory pathways, we performed a multiple-variations test (ANOVA).
We first filtered the overall proteome to require that proteins be quantified in at least 4 out of 6 replicates in one of the groups, which led to 1,520 high quality proteins. The ANOVA analysis resulted in 282 significant proteins among the three groups (Permutation FDR 0.05) ( Figure 2D, Supplementary Table S2), which was further verified by their distinct segregation in a PCA plot ( Figure 2C). Gene Ontology (GO) enrichment analysis indicated that these proteins were generally involved in the oxidation-reduction process, metabolic process, and fatty acid betaoxidation. While acute-phase response proteins and proteins that are associated with the immune system process were largely increased upon septic injury, oxidation-reduction, as well as metabolic process proteins, showed varied profiles ( Figure 2E). This data highlighted the complex and dynamic host response to the septic infection. By examining the subcellular localization of the ANOVA significant proteins, we found that nearly 44% of them (124 out of 282) were enriched in "mitochondrion" (p = 8.14 x 10 -57 ) (Supplemental Figure S2). Of note, around 21% of mitochondrial-associated proteins were identified in a global kidney proteome 34 , and less than 10% in the overall mouse proteome (17,034 Reviewed UniProt proteins). Among the mitochondrial proteins, 46 (37%) of them were annotated as mitochondrial inner membrane proteins, such as ATP synthase (e.g., Atp5j, Atp5l, and Atp5o), NADH dehydrogenase (e.g., Ndufb1/3/4/7/9/12/13) and cytochrome c oxidase (e.g., Cox2 and Cox4i1). This finding is consistent with the biological process enrichment result, and suggests that mitochondrial dysfunction is strongly associated with the decline of kidney integrity and hemostasis 35 , and in our case, during sepsis 36 .

Septic injury induces dynamic changes in kidney phosphoproteome.
To gain more insights into the temporal regulation and functional changes of kidney proteins during S-AKI, we performed phosphoproteome analysis to the same set of tissue samples used for the global proteome by guest on September 23, 2020 analysis (as shown in Figure 2A). In total, 4,745 phosphosites were identified from our study, of which 2,952 were unambiguously localized with high confidence (localization score > 0.75), corresponding to 1,279 non-redundant phosphoproteins (Supplementary Table S3 and S8), which were used for downstream analysis. Of note, phosphorylation of Erk-1/Mapk3 (at Tyr205) and ASC (at Ser193), as described in the previous section, were both seen in our phosphoproteome dataset. However, phosphoproteomic data did not correlate quite well with immunoblot assays (Supplemental Figure S3), which was likely due to the low abundance of these phosphoproteins. Majority (64%) of the 1,279 proteins were exclusively identified from the phosphoproteomics experiments (Supplemental Figure S4A), implying the specificity of the TiO2-based phosphorylation enrichment process. In the context of the reproducibility of phosphoproteome measurements, a much lower correlation was seen both between biological replicates (average R 2 = 0.82) and different groups (average R 2 = 0.79) (Supplemental Figure   S4B) as compared to those of global proteomes. These data suggest that different phosphorylation patterns exist, as indicated by a previous tissue phosphoproteome study 37 , and temporal phosphoproteomic changes in response to microbial infection are more dynamic than global proteomic changes.
To base our analyses on high quality data, we filtered the phosphorylation data set to exclude samples that contained less than 1,000 quantifiable phosphosites, and phosphosites that were quantified in less than 3 replicates, which resulted in 1,870 phosphosites. Subsequent ANOVA analysis resulted in 370 significant phosphosites among the three groups (p < 0.05) Table S4), and further p-value correction (Permutation FDR 0.05) resulted in 23 phosphosites, as presented in Figure 3A. Overall, the majority of the kidney phosphorylation phosphoproteins associated with RNA splicing and mRNA processing were significantly overrepresented (p < 10 -15 ) ( Figure 3B). These include RNA binding motif (RBM) protein families (e.g., Rbm10/17/25/39/xl1), serine/arginine-rich splicing factors (e.g., Srsf1/9) and serine/arginine repetitive matrix proteins (e.g. Srrm1/2). Alternative RNA splicing regulates host immune response in a variety of viral and bacterial infection conditions 38 , and RBM proteins have been found to modulate apoptosis during infection in addition to splicing 39  elegans, its mutation significantly reduced mitochondrial energy metabolism and increased oxidative stress 43 , and previous studies have demonstrated that it is strongly linked to the genetic disease propionic acidemia 43 . Phosphorylation of Pcca at site S248 has been identified by by guest on September 23, 2020 proteomic screening from various mouse tissues previously 44,45 , however, the phosphorylation/dephosphorylation of this site in response to polymicrobial infections and its association with mitochondrial metabolism in kidneys is unknown.
Another interesting class of proteins that was identified in our analyses is the solute carrier (SLC) proteins, a superfamily of membrane-bound transporters with nearly 400 members 46 . They regulate ion transport, waste removal, and many other essential physiological functions, and have increasingly been targeted for therapeutic invention 47 . However, their involvement in septic injury has not been well studied. In our global and phosphoproteomic studies, 72 SLC proteins as well as 113 phosphosites were identified (Supplemental Table S1 and S3). The majority of SLC proteins identified in our study did not show significant differences upon septic injury, and even if changed, the fold differences were minimal ( Figure 4B). The significantly (ANOVA, p < 0.05) changed SLC proteins and/or their phosphorylated forms are shown in Figure 4A. The Band 3 anion transport protein (Slc4a1) increased dramatically (FC > 4) during acute septic infection at day 2, whereas its phosphorylated form (pS18) did not change ( Figure 4C). On the other hand, the unmodified form of the solute carrier family 22 member 12 (Slc22a12, also known as Urat1) did not show significant alterations, but its phosphorylation at S534 was maintained at a similar level at day 2 and then down-regulated at day 7 ( Figure 4D). Phosphorylation of Slc22a12 has been shown to assist in the reabsorption of uric acid via other organic anion transporters 48,49 .
Lastly, both the unmodified and phosphorylated forms of the solute carrier family 22 member 2 (Slc22a2) were changed in a similar trend after septic injury ( Figure 4E). These data indicate that the homeostasis of ions and small molecules in the kidney are broadly altered after sepsis induction.
Discovery-based studies recapitulate signature proteins for S-AKI prognosis and disease progression. Next, we sought to examine signature proteins that correlate with the severity of S-AKI. We performed pairwise comparisons between control and septic kidneys at day 2 or day 7 by guest on September 23, 2020 to highlight significant marker proteins (Figure 5, A-B; Supplementary Table S5). In our data, the known AKI marker protein NGAL dramatically increased (> 64-fold; p < 0.001) at day 2 and gradually lowered down to near basal level (p > 0.05; Figure 5C). This finding was consistent with the immunoblot analysis (Figure 1, C-D), highlighting the value of our MS-based approach for kidney disease biomarker discovery. In addition to NGAL, we also identified other marker proteins and proteins that have not been previously associated with kidney injury. Hydroxymethylglutaryl-CoA synthase 2 (Hmgcs2), a key enzyme of ketogenesis mediating energy generation from lipids when carbohydrates are deprived, was increased in day-2 kidneys by more than 80-fold ( Figure   5D). This data was confirmed by immunoblot analysis (Figure 5, M and N). Hmgcs2 has been indicated to be up-regulated in both Type II diabetic kidneys and Type I diabetic heart 50,51 , suggesting enhanced ketone body production and activated ketogenesis in these diseased organs. Serine protease inhibitor A3N (Serpina3n) was also significantly increased at day 2 by nearly 60-fold ( Figure 5E). Serine protease inhibitors are usually found to be increased during acute inflammation and act by modulating protease activities 52,53 . Recently, Serpina3n was found to be elevated in the urine of rats with early AKI-to-CKD transition and relocated from cytoplasm to apical tubular cell membranes 54 . In our immunoblot analysis, we found one of these serine protease inhibitors, α-1-antitrypsin, to be significantly increased at day 2 ( Figure 5, M and N).
Other proteins such as serum amyloid A-1 and A-2 protein (Saa1 and Saa2) and serum amyloid P-component (ApcS), proteins that were shown to be increased in diabetic kidneys and chronic renal failure 55,56 , were also markedly increased in day-2 post-CLP kidneys (Figure 5, F-H).
Of note, a number of proteins showed consistent up-regulation at both day 2 and day 7 post-CLP. Ceruloplasmin (Cp) is a copper-containing ferroxidase that oxidizes ferrous iron (Fe 2+ ) to its nontoxic ferric iron (Fe 3+ ) form. Its expression was shown to be increased in aging mice or mice consuming high-calorie diet 57 , suggesting the increased amount of oxidative damage in these mice required enhancement for its antioxidant activity. The urinary Cp level was suggested by guest on September 23, 2020 to be a biomarker for CKD in sickle cell disease patients 58 . Cp levels in our day-2 and day-7 mice post-CLP were elevated by 32-and 15-fold, respectively (Figure 5I), suggesting increased oxidative stress in kidneys post-sepsis. Meanwhile, S100a8 and S100a9, DAMPs molecules that mediate pro-inflammatory responses via TLR4 binding 59 , were previously indicated to be increased in plasma and associated with septic shock 60 . In our proteome analysis, the increase was significant at both time points for S100a9, whereas it was significant only at day 7 for S100a8 ( Figure 5, K-L), suggesting the formation of the active dimerized calprotectin was gradually increased in kidneys after septic injury 59 . Regarding the validation assays, Western blot confirmed the differential expression pattern of S100a8, whereas contradicted S100a9 expression ( Figure 5, M-N). The role of the latter in septic kidney would require further investigation.
Among the proteins that were significantly increased post-CLP, we found the upregulation of chitinase-like protein 3 (Chil3) to be most striking, by almost 120-fold at day 2 and more than 150-fold at day 7 ( Figure 5J). These differences were validated with our immunoblot analysis ( Figure 5, M and N). Chil3 is a member of the mammalian chitinase family that lacks the chitinase activity though it is highly secreted by macrophages and neutrophils 61,62 . Similar to the function of S100 proteins, chitinase-like proteins have also been shown to be mediators of inflammation and antimicrobial responses in multiple models of disease such as asthma and pulmonary fibrosis as well as involved in tissue remodeling and wound healing [63][64][65] . In previous work in kidney disease models, a urinary proteomic study has shown that chitinases-like proteins could be candidate biomarkers for S-AKI 65 . Recently, it has been demonstrated to be largely increased in urine from clinical AKI patients 66 . Another study also showed renal Chil3 to be up-regulated at 6 hours and 24 hours after intraperitoneal injection of LPS into mice 67 . Taken together, our study recapitulated previously described sepsis-related modulator proteins and provided a valuable resource of candidate targets to future studies of sepsis diagnosis and therapeutics.
by guest on September 23, 2020 Molecular-level understanding of AKI-CKD transition. As described above, our data have shown distinct pathology of the septic kidneys at the acute and chronic stages by analyzing them at day 2 and day 7 post-septic injury, respectively. Therefore, a direct comparison between these two phases would lead to insights into how the disease progresses after acute septic injury.
Following a similar t-test (FC ≥ 1.5; Permutation FDR 0.05), we identified 38 and 56 up-and downregulated proteins, respectively, from day-7 mice in comparison to day-2 mice (Supplementary Table S6). Interestingly, although the functional enrichment analysis did not suggest particular biological process terms enriched among the up-regulated proteins, likely due to the low number of input proteins, we noticed several immunoglobulin proteins (such as Ighm, Ighg, Igkc, IGHG3, and HVM51) were significantly increased. This suggested that while recovering from the acute phase of septic-induced kidney injury, there may be an initiation of an adaptive immune response in renal tissue progressing toward chronic kidney disease. Indeed, a switch from innate to adaptive immunity has been reported to mediate the transition from acute septic kidney injury to prolonged chronic disease 68,69 , and some of these immunoglobulins can be used to develop biomarkers for CKD in the future 70 .
To obtain a broader view of the dysregulated pathways during S-AKI-CKD transition, we slightly lowered the fold-change threshold (≥ 1.2) to capture more differential proteins (202 in total; Figure 6A) and build a signaling network (see details in Experimental Procedures). The analysis with the highest confidence score (cutoff = 0.9) resulted in 396 interactions and 126 nodes ( Figure   6B), the majority (56%) of which were associated with small-molecule metabolic processes (FDR = 3.35e-48), such as lipids, nucleotides, alcohol, and other fatty acids. Abnormal lipid metabolism has been associated with diabetic nephropathy and other kidney dysfunctions 71,72 ; lipid deposition contributes to atherosclerosis and cardiovascular diseases commonly seen in CKD patients 73 .
The network analysis also indicated several interesting protein clusters such as oxidoreductase complex, peroxisome, and cytochrome P450 (CYP) family proteins (Figure 6B), which may play  75,76 . In our analysis, five CYP members (2A4, 2A5, 2E1, 4A10, and 4A14) were found to be significantly down-regulated in day-7 kidneys compared to day-2. For example, the expression of Cyp4a10 was dramatically increased after acute infection (at day 2) and declined back to the basal level at chronic stage (at day 7) (Figure 6C). It has been noted that CYP enzymes can be selectively regulated in different states of inflammation by a multitude of cytokines released after infection or inflammation 75 . In hepatic and extrahepatic tissues, chronic inflammation is usually associated with downregulation of CYP enzymes 75 . Hence, our quantitative data of CYP proteins were in line with previous studies and suggest that they could also be used as signatures for kidney disease severity and/or classification. Notably, carboxylesterase 1C (Ces1c), which aids in detoxification of xenobiotics and is responsible for poor pharmacokinetics of the anti-virus drug remdesivir 77 , was also mapped in our cluster analysis. Ces1c has been shown to be significantly decreased in macrophages after LPS treatment 78 , which was consistent with its changes in our data of septic kidneys ( Figure 6D).
Together, our proteomic data provides an unbiased view of how kidney tissues dynamically modulate oxidative stress caused by sepsis, and shed light on future exploration for potential diagnostics and therapeutics.

Discussion
by guest on September 23, 2020 In the present study, we have profiled the septic kidney proteome and phosphoproteome using a CLP mouse model established in our laboratory. CLP was performed in six mice (biological replicates) in each of three groups to better control reproducibility. The tissue samples were processed following an STrap approach developed by Zougman and colleagues 79 . The STrap filters were in-house packed using bulk glass fiber membranes, which are nearly cost-free and as effective as commercial filters 19 . The sample was analyzed in a 'single-shot' injection with label-free quantitation based on MaxLFQ algorithm. Our data showed minimal technical variations, thus offering critical insights into the molecular level alterations of renal proteins upon septic injury.
Our CLP procedure induced only low to mid-grade severity and nearly all of the day-7 mice survived from the acute sepsis and progressed to the chronic stage. Hence, the CLP model allowed us to investigate not only the acute host response (at day 2) but also the "acute-to-chronic" transition even after moderate kidney injury, the latter of which has not been well defined molecularly 80,81 . This transition from the acute to chronic stage reflects the refractoriness of septic kidney diseases in the clinical setting. Our study provides solid molecular evidence for the first time derived directly from septic kidneys.
S-AKI can occur in the absence of overt signs of hypoperfusion or clinical signs of hemodynamic instability. Therefore, early biomarkers are urgently needed to permit timely diagnosis and predict injury severity 82 . However, sepsis is usually associated with multiorgan injuries, and traditional markers of S-AKI originated from plasma and urine (e.g., creatinine, urinary concentrating ability) are susceptible to interference from non-renal factors, thus are not sensitive and specific 82 . Our global proteome and phosphoproteome data broadly agree with previous urinary discoveries in the context of many known AKI markers such as NGAL. In addition, we validated several new and/or less-well studied marker proteins for S-AKI including Hmgcs2, Serpin, S100a8, and Chil3. The comprehensive global analyses revealed that at the AKI stage, immune-related proteins were up-regulated while metabolism-related proteins were repressed in by guest on September 23, 2020 septic kidneys. Our data suggests that multiple immune pathways could be activated in kidneys after septic injury, including adaptive DAMPs responses, CYP-mediated lipid metabolism, peroxisome-mediated oxidative stress, and likely the inflammasome-mediated pyroptosis pathway. Effector molecules involved in the canonical inflammasome formation and pyroptosis activation [83][84][85] , including GSDMD, cleaved caspase-1, and phosphorylated-ASC, were all found to be significantly increased post-sepsis. Gsdmd-dependent pyroptosis cell death has been revealed for the first time in non-septic AKI in a very recent study 32 . Our data indicated that Gsdmd expression remained at a low level at the acute stage and drastically increased at the chronic phase. Adding another time point midway through this transition (for instance, at day 4 or day 5) would likely reveal the onset of pyroptosis, which can be considered in the future studies.
Nevertheless, our findings implied the potential role of pyroptosis in initiating tubular cell damage, thus deteriorating CKD, or alternatively, plasma membrane repair can be initiated to counterbalance pyroptosis (for instance, through ESCRT 86,87 ). These questions are certainly of great interest for future investigations.
Among the responsive proteins to septic injury, Chil3 was found to be of particular interest.
Chil3 (also known as YKL-40 in humans and BRP-39 in mice) does not contain known chitinase activity, though it has been implicated in host defense, inflammation, and remodeling processes 63,64 . Chil3 has been reported as a biomarker for skin wound infection 88 , cardiovascular disease and diabetes 89 , and cancer 90 . A handful of studies have shown that Chil3 has critical functional relevance to kidney ischemic injury 91 and renal fibrosis 92 . There is also evidence showing its potential in predicting the onset and recovery of acute kidney injuries 65,92,93 . However, the majority of these studies investigated chitinase secretion into the urine and used the urinary form as an indicator of disease status, which might be complicated by other non-kidney diseases such as urothelial carcinoma 20 . Emerging studies have also started to demonstrate the mechanism of Chil3 in mediating immunity, including its role in IL-17 production in γδ T cells 94 , and recruitment by guest on September 23, 2020 of macrophages and neutrophils in colorectal cancer 95 . A recent study showed that Chil3 interacts with interleukin-13 receptor α2 and activates pathways that regulate apoptosis, pyroptosis, inflammasome formation, and antibacterial responses 96 . Our data showed that Chil3 has a strong correlation with S-AKI disease onset and severity. Chil3 was elevated almost 120-fold at day 2 and more than 150-fold at day-7 after S-AKI, implying that it was involved in the progression of septic kidney injures. It remains unclear, however, whether the expression Chil3 is linked to the direct microbial migration from gut to kidney.
Furthermore, plenty of metabolism-related proteins were significantly changed during the acute septic injury stage, which is consistent with previous findings that changes in metabolism and mitochondrial energetics occurred in response to renal damage 97,98 . Of note, the general reduction of phosphorylation after septic injury indicates that energy usage has been reprioritized in renal tissue. The drastic increase of Hmgcs2 at day 2 suggests that ketogenesis is highly upregulated during acute kidney injury. Similar up-regulation of Hmgc2 in diabetic kidney and heart encourages us to speculate that the energy-producing pathway in kidneys during S-AKI may be ketogenesis. Therefore, we can imagine that our CLP procedure in this study merely generated a moderate S-AKI model, and it remains intriguing to see if, for example, during a severe S-AKI scenario, or at an earlier time point post-CLP, more drastic changes of these marker proteins will be seen.
Since cecal perforation breaks intestinal barrier, and leads to the translocation of fecal contents from gut lumina into the peritoneum and subsequently blood stream 99 , we have attempted to detect gut microbial species, if they might have translocated to distal organs such as kidney. Metaproteomic database searches were performed to identify potential bacterial proteins in addition to the mouse host proteins. Although not totally unexpected, we did not obtain highly confident protein hits, as they were higher than the 1% FDR threshold and also majority matched with single peptides (Supplemental Table S7). We did not intent to over interpret the by guest on September 23, 2020 data as our study used a mid-severity CLP murine model, gut microbes might be cleared already by the first line of host immunity before gaining access to distal tissues or organs, thus minimizing the chance of identifying them in the complex background of kidney tissue proteins. In addition, the histological data of kidney tissue did not show clear evidence of microbial presence neither ( Figure 1E). Indeed, gut bacterial DNA fragments have been detected in the blood of CKD patients 100,101 . Gut-originated uropathogenic Escherichia coli has also been reported to be able to replicate intracellularly in bladder urothelium, causing recurrent urinary tract infections and cystitis 102,103 . In addition, emerging evidence suggested that respiratory tract extracellular pathogen Streptococcus pneumoniae could translocate to heart tissue and replicate intracellularly, exacerbating community-acquired pneumonia by inducing cardiac microlesions 104,105 . Currently, a prevailing paradigm in renal physiology is the assumption that the kidney is sterile 106 . Whether gut microbes can invade and colonize kidney is still an unanswered question. Our data from the current study did not provide strong evidence of microbial presence in kidney, however, we hypothesize that gut microbes, once release to circulation system, may gain access to other tissues, form local bacterial colonization, then amplify existing injury and lead to persistent injuries.
Therefore, further investigations (for instance, using severe CLP model) are desired to pinpoint the potential role of gut microbes in the chronic kidney diseases.
Our study is not flawless. One of the apparent limitations is the relatively low coverage of the kidney proteome and phosphoproteome. With further fractionation prior to LC-MS/MS analysis, the sample complexity would be reduced and the chance to identify marker proteins that have low abundance would be increased. We have decided to demonstrate that the single-shot proteomics along with the extremely economical sample preparation approach would be a great alternative to biomarker discovery and pathology mechanism investigation. In summary, our study provides the first characterization of how moderate gut injury triggered sepsis promotes S-AKI and the molecular determinant alterations in situ. The findings from our study will facilitate our     App in Cytoscape software. The score cutoff for interaction confidence was set to 0.9. Shaded clusters are involved in metabolic processes. The color coding is in accordance to fold changes between day 7 and day 2. Diamond shape depicts mitochondrion associated proteins. (C-D) Intensity plot of two representative proteins associated with the oxidation-reduction process.
by guest on September 23, 2020