The proteome microenvironment determines the protective effect of preconditioning in cisplatin-induced acute kidney injury

Acute kidney injury (AKI) leads to significant morbidity and mortality; unfortunately, strategies to prevent or treat AKI are lacking. In recent years, several preconditioning protocols have been shown to be effective in inducing organ protection in rodent models. Here, we characterized two of these interventions—caloric restriction and hypoxic preconditioning—in a mouse model of cisplatin-induced AKI and investigated the underlying mechanisms by acquisition of multi-layered omic data (transcriptome, proteome, N-degradome) and functional parameters in the same animals. Both preconditioning protocols markedly ameliorated cisplatin-induced loss of kidney function, and caloric restriction also induced lipid synthesis. Bioinformatic analysis revealed mRNA-independent proteome alterations affecting the extracellular space, mitochondria, and transporters. Interestingly, our analyses revealed a strong dissociation of protein and RNA expression after cisplatin treatment that showed a strong correlation with the degree of damage. N-degradomic analysis revealed that most posttranscriptional changes were determined by arginine-specific proteolytic processing. This included a characteristic cisplatin-activated complement signature that was prevented by preconditioning. Amyloid and acute-phase proteins within the cortical parenchyma showed a similar response. Extensive analysis of disease-associated molecular patterns suggested that transcription-independent deposition of amyloid P-component serum protein may be a key component in the microenvironmental contribution to kidney damage. This proof-of-principle study provides new insights into the pathogenesis of cisplatin-induced AKI and the molecular mechanisms underlying organ protection by correlating phenotypic and multi-layered omics data. Acute kidney injury (AKI) leads to significant morbidity and mortality; unfortunately, strategies to prevent or treat AKI are lacking. In recent years, several preconditioning protocols have been shown to be effective in inducing organ protection in rodent models. Here, we characterized two of these interventions—caloric restriction and hypoxic preconditioning—in a mouse model of cisplatin-induced AKI and investigated the underlying mechanisms by acquisition of multi-layered omic data (transcriptome, proteome, N-degradome) and functional parameters in the same animals. Both preconditioning protocols markedly ameliorated cisplatin-induced loss of kidney function, and caloric restriction also induced lipid synthesis. Bioinformatic analysis revealed mRNA-independent proteome alterations affecting the extracellular space, mitochondria, and transporters. Interestingly, our analyses revealed a strong dissociation of protein and RNA expression after cisplatin treatment that showed a strong correlation with the degree of damage. N-degradomic analysis revealed that most posttranscriptional changes were determined by arginine-specific proteolytic processing. This included a characteristic cisplatin-activated complement signature that was prevented by preconditioning. Amyloid and acute-phase proteins within the cortical parenchyma showed a similar response. Extensive analysis of disease-associated molecular patterns suggested that transcription-independent deposition of amyloid P-component serum protein may be a key component in the microenvironmental contribution to kidney damage. This proof-of-principle study provides new insights into the pathogenesis of cisplatin-induced AKI and the molecular mechanisms underlying organ protection by correlating phenotypic and multi-layered omics data. Acute kidney injury (AKI) is one of the most common complications in hospitalized patients and shows a rapidly increasing incidence—especially in the elderly.1Hsu R.K. McCulloch C.E. Dudley R.A. et al.Temporal changes in incidence of dialysis-requiring AKI.J Am Soc Nephrol. 2013; 24: 37-42Google Scholar, 2Hsu C.-Y. McCulloch C.E. Fan D. et al.Community-based incidence of acute renal failure.Kidney Int. 2007; 72: 208-212Google Scholar Although partial recovery occurs in the majority of patients, AKI is strongly associated with prolonged hospitalization, increase in mortality, and progression to end-stage renal disease.3Wald R. Quinn R.R. Luo J. et al.Chronic dialysis and death among survivors of acute kidney injury requiring dialysis.JAMA. 2009; 302: 1179-1185Google Scholar, 4Thakar C.V. Christianson A. Himmelfarb J. et al.Acute kidney injury episodes and chronic kidney disease risk in diabetes mellitus.Clin J Am Soc Nephrol. 2011; 6: 2567-2572Google Scholar, 5Lafrance J.-P. Miller D.R. Acute kidney injury associates with increased long-term mortality.J Am Soc Nephrol. 2010; 21: 345-352Google Scholar, 6Prendecki M. Blacker E. Sadeghi-Alavijeh O. et al.Improving outcomes in patients with acute kidney injury: the impact of hospital based automated AKI alerts.Postgrad Med J. 2016; 92: 9-13Google Scholar, 7Chertow G.M. Burdick E. Honour M. et al.Acute kidney injury, mortality, length of stay, and costs in hospitalized patients.J Am Soc Nephrol. 2005; 16: 3365-3370Google Scholar End-stage renal disease can also be the consequence of recurrent minor episodes of AKI during lifetime.8Dear J.W. Yuen P.S.T. Setting the stage for acute-on-chronic kidney injury.Kidney Int. 2008; 74: 7-9Google Scholar AKI is associated with financial burden to modern health care systems exceeding an estimate of $10 billion per year in the United States.7Chertow G.M. Burdick E. Honour M. et al.Acute kidney injury, mortality, length of stay, and costs in hospitalized patients.J Am Soc Nephrol. 2005; 16: 3365-3370Google Scholar, 9Silver S.A. Long J. Zheng Y. et al.Cost of acute kidney injury in hospitalized patients.J Hosp Med. 2017; 12: 70-76Google Scholar, 10Hobson C. Ozrazgat-Baslanti T. Kuxhausen A. et al.Cost and mortality associated with postoperative acute kidney injury.Ann Surg. 2015; 261: 1207-1214Google Scholar Although pre- or postrenal AKI can be detected and treated rather readily, the actual cellular damage of kidney tissue (intrarenal AKI) is the major determinant of outcome. Several key factors causing intrarenal AKI are known; among these are ischemia-reperfusion during injury surgery and nephrotoxic agent exposure. However, targeted ways to prevent this damage are still not available. In 1986 Murry et al. showed that the exposure of the myocardium to sublethal levels of ischemia can protect animals from consecutive myocardial infarction—a concept called ischemic preconditioning.11Murry C.E. Jennings R.B. Reimer K.A. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium.Circulation. 1986; 74: 1124-1136Google Scholar This effect is not limited to the heart but can also be exploited in other organs such as the kidney.12Cochrane J. Williams B.T. Banerjee A. et al.Ischemic preconditioning attenuates functional, metabolic, and morphologic injury from ischemic acute renal failure in the rat.Ren Fail. 1999; 21: 135-145Google Scholar Since then, several renal preconditioning interventions have been characterized primarily in rodent models. Two prominent examples are caloric restriction (CR) and hypoxic preconditioning (HP) that showed substantial protective potential for AKI.13Mitchell J.R. Verweij M. Brand K. et al.Short-term dietary restriction and fasting precondition against ischemia reperfusion injury in mice.Aging Cell. 2010; 9: 40-53Google Scholar, 14Bernhardt W.M. Câmpean V. Kany S. et al.Preconditional activation of hypoxia-inducible factors ameliorates ischemic acute renal failure.J Am Soc Nephrol. 2006; 17: 1970-1978Google Scholar, 15Yang C.-C. Lin L.-C. Wu M.-S. et al.Repetitive hypoxic preconditioning attenuates renal ischemia/reperfusion induced oxidative injury via upregulating HIF-1α-dependent bcl-2 signaling.Transplantation. 2009; 88: 1251-1260Google Scholar Unfortunately, these strategies have not made their way to clinical application so far because of problems with feasibility on the one hand and reproducibility in the much more complex setting of patients’ care on the other hand. Consequently, a more profound knowledge of the molecular key changes in different forms of AKI and the molecular mechanisms underlying preconditioning-mediated organoprotection is essential to uncover refined therapeutic targets in patients. Here, we demonstrate that both HP and CR can be used to prevent intrarenal damage inflicted by nephrotoxic treatment in mice. We then applied multilayered omics approach to investigate the molecular composition of a protected damaged kidney versus an unprotected damaged kidney to prioritize the most important molecular patterns associated with renal damage. Transcriptomic (mRNA sequencing) and proteomic analyses revealed a strong dissociation of protein and RNA expression after AKI, correlating with the severity of injury. N-Degradomic analyses exposed that most posttranscriptional changes were determined by serine protease-dependent proteolytic processing, including a characteristic complement activation signature. To induce AKI we applied an i.p. bolus of cisplatin to untreated wild-type mice (Figure 1a ). At the same time, we administered cisplatin to mice either preconditioned by CR13Mitchell J.R. Verweij M. Brand K. et al.Short-term dietary restriction and fasting precondition against ischemia reperfusion injury in mice.Aging Cell. 2010; 9: 40-53Google Scholar (only 66% of the average daily intake of calories for 28 days) or by incremental hypoxia16Bernaudin M. Nedelec A.S. Divoux D. et al.Normobaric hypoxia induces tolerance to focal permanent cerebral ischemia in association with an increased expression of hypoxia-inducible factor-1 and its target genes, erythropoietin and VEGF, in the adult mouse brain.J Cereb Blood Flow Metab. 2002; 22: 393-403Google Scholar (HP; reduction of oxygen to 8.3% in a hypoxia chamber for 2, 4, and 8 hours on 3 consecutive days) (Figure 1a). Compared with vehicle-treated (veh) mice, cisplatin-treated (cis) mice showed a significant weight loss after 72 hours (1-way analysis of variance and Bonferroni multiple comparison test, P < 0.001). Calorically restricted (CR) and hypoxia-treated (HP) animals did not lose weight significantly 72 hours after AKI (1-way analysis of variance and Bonferroni multiple comparison test, P < 0.05) (Figure 1b; Supplementary Figure S1). Consistent with a protective effect of our preconditioning strategies, blood urea nitrogen (BUN) and plasma creatinine levels were significantly higher in non-preconditioned animals after cisplatin administration than in preconditioned animals (Figure 1c and d). Plasma values of creatinine and BUN showed a strong correlation with plasma values of cystatin C in a different cohort, supporting the loss of kidney function due to cisplatin treatment and prevention by preconditioning (Supplementary Figure S2). Histologically, kidneys of cis control mice showed a significant deposition of periodic acid-Schiff (PAS)–positive material in the interstitium (Figure 1e). In addition, nuclear pyknosis indicative of cell death and intratubular protein casts were observed in the kidneys treated with cisplatin. In HP animals, kidneys showed a significantly better histological appearance with less nuclear pyknosis and less casts. CR mice showed a histological outcome after cisplatin treatment that was not notably different from kidneys obtained from veh animals (Figure 1e). Semiquantitative analysis of histological damage showed a protective effect, particularly in CR animals (Supplementary Figure S3A). To further validate histological damage, we performed immunostaining of kidney injury molecule-1 (KIM1), a marker for renal damage,17Moledina D.G. Parikh C.R. Phenotyping of acute kidney injury: beyond serum creatinine.Semin Nephrol. 2018; 38: 3-11Google Scholar which suggested a stronger renal damage in cis > HP > CR > veh (Supplementary Figure S3B). Analysis of regulated cell death as determined by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling assay (TUNEL) demonstrated a high number of positive cells in the kidneys of animals treated with only cisplatin, significantly less after HP and none in CR or veh animals (Figure 1f). These results demonstrate that preconditioning interventions have the potential to ameliorate cisplatin-induced intrarenal damage. To characterize the molecular changes induced by cisplatin toxicity and their modulation by preconditioning, we performed quantitative label-free proteomic analysis of the renal cortices of the same mice we used for the analyses mentioned above and identified >6000 proteins; 4733 proteins could be identified across all samples (see Methods for details; see Supplementary Table S1 for detailed results). Using hierarchical clustering for Euclidean distance, we found a clear separation between veh and cis kidneys (Figure 2a ). We also found that in principal component analysis, cis and veh samples were clearly distinguishable. In contrast, proteomic profiles of veh and cis kidneys after CR were similar (Figure 2b). Then, we analyzed the effect of cisplatin on kidney tissue as compared to vehicle treatment. A comparison of protein abundance between both conditions with stringent parameters (s0 = 0.1 and false discovery rate [FDR] < 0.01) identified 1448 significantly changed proteins; 636 proteins were increased and 812 were decreased after cisplatin treatment (Figure 2c). We found many proteins known to increase with AKI; among these were neutrophil gelatinase-associated lipocalin (gene symbol: Lcn218Ko G.J. Grigoryev D.N. Linfert D. et al.Transcriptional analysis of kidneys during repair from AKI reveals possible roles for NGAL and KIM-1 as biomarkers of AKI-to-CKD transition.Am J Physiol Renal Physiol. 2010; 298: F1472-F1483Google Scholar) and Fetuin-A (gene symbol: Ahsg19Zhou H. Pisitkun T. Aponte A. et al.Exosomal Fetuin-A identified by proteomics: a novel urinary biomarker for detecting acute kidney injury.Kidney Int. 2006; 70: 1847-1857Google Scholar). A gene ontology term enrichment (FDR = 0.02) analysis revealed that proteins related to the extracellular matrix and acute phase reaction were increased after cisplatin exposure, consistent with an inflammatory and fibrotic response in the tissue (Figure 2d). Moreover, proteins associated with transmembrane receptor protein kinase activity were increased, consistent with protein alterations affecting intracellular response machineries (Figure 2d). Conversely, proteins associated with the excretion of ions, amino acids, and metabolites were decreased in the data set, as well as proteins associated with the mitochondria (Figure 2d). Extracellular matrix is an ambiguous term in which multiple biological entities are lumped together (e.g., secreted proteins and known basal lamina and “matrisome”-associated proteins).20Naba A. Pearce O.M.T. Del Rosario A. et al.Characterization of the extracellular matrix of normal and diseased tissues using proteomics.J Proteome Res. 2017; 16: 3083-3091Google Scholar, 21Hobeika L. Barati M.T. Caster D.J. et al.Characterization of glomerular extracellular matrix by proteomic analysis of laser-captured microdissected glomeruli.Kidney Int. 2017; 91: 501-511Google Scholar Among the upregulated extracellular proteins, we found core matrisome proteins, but also secreted matrisome-associated proteins as well as secreted proteins not related to the matrisome (e.g., amylase and lysozyme) (Figure 2e). Among these secreted proteins, we found an increase in particular smaller proteins that are more easily filtered and potentially taken up by endocytic mechanisms in the proximal tubule22Grahammer F. Ramakrishnan S.K. Rinschen M.M. et al.mTOR regulates endocytosis and nutrient transport in proximal tubular cells.J Am Soc Nephrol. 2017; 28: 230-241Google Scholar (Figure 2f). Next, we performed hierarchical clustering analyses to define global proteomic shifts across the 4 treatment groups (Figure 3a ). Six major clusters, each with >10 proteins, could be defined (Figure 3b). Three clusters had protein patterns increasing with the extent of damage (protein expression veh < CR < HP < cis). These clusters were significantly enriched for several distinct processes such as complement, extracellular matrix generation, and chemotaxis (Figure 3c). One cluster showed a decreased protein expression with the extent of damage (protein expression veh > CR > HP > cis). This cluster was largely enriched for transmembrane proteins and proteins involved in proximal tubule function such as sodium transport and amino acid transport. One cluster containing 13 proteins (cluster 5) peaked specifically in the kidneys of animals of preconditioned by CR, which were almost completely protected from cisplatin-induced damage. This cluster was enriched for proteins involved in fatty acid biosynthesis, indicating that the induction of these proteins may be involved in the protective capacity of CR. To further investigate whether these changes were relevant, we performed targeted lipidomic analysis by mass spectrometry with direct infusion of lipid extracts from treated mouse kidneys in a different cohort. The levels of different glycerophospholipid subspecies (phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, and phosphatidylserines) were determined by lipid class–specific neutral loss and multiple precursor ion scanning (see Supplementary Table S2 for detailed results). This analysis revealed a loss of membrane lipids during cisplatin-induced injury. This loss of lipids was lower after CR, suggesting that a loss of membrane integrity is associated with cisplatin-induced injury, which can be abolished by CR (Figure 3d). Prominent alterations in mRNA expression have been determined by high-throughput analyses of kidneys after the induction of different modes of AKI.23Kumar S. Liu J. McMahon A.P. Defining the acute kidney injury and repair transcriptome.Semin Nephrol. 2014; 34: 404-417Google Scholar Yet, mRNA expression does not necessarily and always predict protein abundance.24Liu Y. Beyer A. Aebersold R. On the dependency of cellular protein levels on mRNA abundance.Cell. 2016; 165: 535-550Google Scholar Thus, we asked whether the extent of dysregulated proteins was largely due to the expression of altered transcripts. To this end, we performed analyses of the transcriptome from the same kidneys as used for the proteomic analyses using next generation sequencing (see Supplementary Table S3 for detailed results). Using t-Distributed Stochastic Neighbor Embedding clustering of the individual transcript levels, we found that samples of veh mice and samples of cis mice after CR were closely related and both showed similar differences in comparison to the samples of non-preconditioned cis animals, consistent with our proteomic data (Figure 4a ). Of note, the fold changes (e.g., in cis vs. veh animals) were less prominent as determined by proteomic analysis (Supplementary Figure S4A and B). Enrichment analysis determined similar pathways being regulated as compared to the proteome (Supplementary Figure S4C and D). Next, we performed a correlation analysis between mRNA and protein copy number level for each individual protein-mRNA pair. The distribution of mRNA-protein correlation coefficients for every protein-mRNA pair revealed that transcript copy numbers only moderately determined protein abundance in all treated samples (Figure 4b) as commonly observed.26Ruggles K.V. Krug K. Wang X. et al.Methods, tools and current perspectives in proteogenomics.Mol Cell Proteomics. 2017; 16: 959-981Google Scholar Interestingly, when analyzing each treatment group separately, we found that correlation between transcript and proteome levels was highest in CR (Figure 4c) (R = 0.57) and significantly lower in HP (R = 0.54) and much lower in cis (R = 0.47). To examine the relationship of a low correlation coefficient of the transcriptome and proteome and the severity of kidney damage, we plotted the correlation coefficient against the individual creatinine value in each mouse analyzed. Notably, we observed a strong and significant connection between the correlation coefficient R and log2(creatinine level) (R = −0.98) (Figure 4d) that could also be recapitulated by using the BUN values (R = −0.96) (Figure 4e). The connection between the correlation coefficient R and log2(creatinine level) suggested that with increased severity of kidney damage transcriptomic data become less predictive of protein abundance, indicating that posttranscriptional changes must play an important role in response to injury. Posttranslational regulatory mechanisms, such as proteolytic cleavage reactions by proteases or the proteasome, have been described as a major regulator of the posttranscriptional landscape in different scenarios of cell death, including AKI.27Galluzzi L. Kepp O. Chan F.K.-M. et al.Necroptosis: mechanisms and relevance to disease.Annu Rev Pathol. 2017; 12: 103-130Google Scholar, 28Kaushal G.P. Singh A.B. Shah S.V. Identification of gene family of caspases in rat kidney and altered expression in ischemia-reperfusion injury.Am J Physiol. 1998; 274: F587-F595Google Scholar Mass spectrometry–based N-degradomics enable the identification and quantification of protein N-termini and proteolytic activity on a proteome-wide scale.29Huesgen P.F. Lange P.F. Overall C.M. Ensembles of protein termini and specific proteolytic signatures as candidate biomarkers of disease.Proteomics Clin Appl. 2014; 8: 338-350Google Scholar, 30Huesgen P.F. Lange P.F. Rogers L.D. et al.LysargiNase mirrors trypsin for protein C-terminal and methylation-site identification.Nat Methods. 2015; 12: 55-58Google Scholar, 31Rinschen M.M. Hoppe A.-K. Grahammer F. et al.N-Degradomic analysis reveals a proteolytic network processing the podocyte cytoskeleton.J Am Soc Nephrol. 2017; 28: 2867-2878Google Scholar We used the established terminal amine isotope labeling of substrates32Kleifeld O. Doucet A. auf dem Keller U. et al.Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products.Nat Biotechnol. 2010; 28: 281-288Google Scholar, 33Rosin D.L. Okusa M.D. Dangers within: DAMP responses to damage and cell death in kidney disease.J Am Soc Nephrol. 2011; 22: 416-425Google Scholar protocol to perform an N-degradomic analysis of the kidneys of the same animals used for proteomic and transcriptomic characterization as described above. The terminal amine isotope labeling of substrates protocol indiscriminately enriches all protein N-terminal peptides including those generated by proteases or those blocked by endogenous N-terminal acetylation. In total, we identified 1865 N-termini in 2 complementary triplex labeling experiments that probed altered cisplatin-induced processing and the protective effect of CR (by comparing cis, CR, and veh; n = 3; 1 experiment was excluded from data analysis because of an error during sample preparation) and HP (by comparing cis, HP, and veh; n = 4) (see Supplementary Figure S5A and Supplementary Table S4 for detailed results). The cis/veh ratios of both experiments correlated well, indicating technical reproducibility (Supplementary Figure S5B). Positional annotation discriminated 1306 “expected” N-termini (matching to position 1 of the predicted protein models or known processing sites such as N-terminal methionine excision; removal of predicted signal-, transit-, or pro-peptide sequences) from 559 N-termini mapping to “unexpected” sites within the corresponding protein models, mostly representing dimethylated protease–generated neo-N-termini (Figure 5a ). Ratiometric analysis of the different conditions in the data set found that cisplatin treatment induced a skewed ratio distribution, particularly among unexpected N-termini, with similar results for kidneys from CR (cis/CR and cis/veh, Figure 5a) and HP (cis/HP and cis/veh, Figure 5b) pretreated mice, indicating increased proteolytic activity in damaged kidneys. Analysis of positional amino acid frequency matrices (motifs) can be used to infer dominant proteolytic activities.31Rinschen M.M. Hoppe A.-K. Grahammer F. et al.N-Degradomic analysis reveals a proteolytic network processing the podocyte cytoskeleton.J Am Soc Nephrol. 2017; 28: 2867-2878Google Scholar Here, motif analysis showed that in the CR experiment, cisplatin treatment mainly increased termini starting with S or T, predominantly resulting from cleavage after a basic residue (K, R, and H, Figure 5c). This was also found in the HP data set (Figure 5d), suggesting a cisplatin-induced activity of arginine- and lysine-specific proteases such as trypsin-like serine proteases. To test whether these alterations were independent of total proteome changes, we tested whether total proteome fold changes in cisplatin-induced damage correlated with the fold change at the respective cleavage sites within the protein. We observed a strong and significant correlation between proteome expression and N-termini abundance (Figure 5e), indicating that cleaved fragment abundance also largely reflected protein abundance. Consistently, we found that there was a weak negative correlation between N-termini abundance and the protein-mRNA correlation coefficient described above (Supplementary Figure S5C). Interestingly, we observed that several termini that were increased in both pretreatment conditions were chiefly extracellular proteins, suggesting that the majority of the observed proteolytic processes induced by cisplatin treatment occurred extracellularly (Figure 5f). We also analyzed proteasome activity as chymotrypsin-like activity in the kidney cortex. There was no significant difference in proteasome activity between the samples (Supplementary Figure S6). This suggests that the observed degradomic alterations are not part of a general unspecific response-regulating proteolytic protein decay, but rather part of specific extracellular proteolytic signaling processes. Given the predominant increase in extracellular processes (Figure 2d), we correlated the individual proteins obtained from the proteomic data with the parameters of kidney function (creatinine/BUN level), revealing a broad distribution of correlation coefficients for individual proteins (Figure 6a ). Reactome term enrichment analysis revealed a strong correlation of the creatinine level with proteins related to extracellular matrix synthesis, serum proteins, and complement activation (Figure 6b). Complementary and consistent with these findings, N-degradomic analysis showed a strong proteolytic activation of the complement cascade demonstrating specific cleavage of complement components C3 and C4 as well as activation of upstream regulators of the lectin-binding, alternative, and classical pathways (Figure 6c; Supplementary Table S5). Finally, we aimed to expand our data set to unknown disease processes. Damage-associated molecular patterns (DAMPs) have been suggested to correspond to acute cell death and tissue damage.33Rosin D.L. Okusa M.D. Dangers within: DAMP responses to damage and cell death in kidney disease.J Am Soc Nephrol. 2011; 22: 416-425Google Scholar Therefore, we analyzed our proteomic data set, which clearly reflects these damage processes, to determine novel patterns of protein-based DAMPs. However, of 72 known DAMPs compiled from the literature (Figure 7a ; Supplementary Table S6), only 27 of them could be identified in the proteomic data set and their mean correlation with the renal damage parameter plasma creatinine level was moderate for the animal cohort (a mean Spearman or Pearson correlation of 0.2). We then expanded the list of known DAMPs to “suggested DAMPs” by inclusion of functionally related protein families in the Reactome database.34Croft D. Mundo A.F. Haw R. et al.The Reactome pathway Knowledgebase.Nucleic Acids Res. 2014; 42: D472-D477Google Scholar Using this strategy, we found that the Reactome pathway “amyloid fiber formation” and especially the serum amyloid P-component (APCS), a circulating plasma protein suggested to be associated with the binding of apoptotic cells and tubule formation in vitro,35Kelly K.J. Kluve-Beckerman B. Dominguez J.H. Acute-phase response protein serum amyloid A stimulates renal tubule formation: studies in vitro and in vivo.Am J Physiol Renal Physiol. 2009; 296: F1355-F1363Google Scholar, 36Pilling D. Buckley C.D. Salmon M. et al.Inhibition of fibrocyte differentiation by serum amyloid P.J. Immunol. 2003; 171: 5537-5546Google Scholar correlated most strongly with the degree of kidney damage (Figure 7b). The correlation at the mRNA level was much less prominent (Figure 7c), and APCS was found to be only moderately increased at the mRNA level (Supplementary Figure S7A). Consistently, accumulation and binding of APCS to tubule structures were also shown in immunohistochemical staining of cis kidneys whereas in veh kidneys only diffuse background staining was shown (Figure 7d). Laser microdissection followed by ultrasensitive mass spectrometry37Höhne M. Frese C.K. Grahammer F. et al.Single-nephron proteomes connect morphology and function in proteinuric kidney disease.Kidney Int. 2018; 93: 1308-1319Google Scholar confirmed that the APCS protein was found in both PAS-positive tubules and PAS-positive protein casts (2 unique peptides) (Supplementary Figure S7B and C). Despite the presence of APCS, a general deposition of amyloid fibers was not detected using Congo red staining and polarized light microscopy (data not shown). Preconditioning-mediated protection from AKI holds the promise to reveal novel targets to prevent AKI in the clinical setting. In this study, we have shown the potential of 2 modes of preconditioning (CR and HP) in the prevention of cisplatin-induced AKI. Cisplatin induced a specific signature on protein abundance with an increased expression of known injury m