International Journal of Molecular Sciences Article The Acute Phase Response Is a Prominent Renal Proteome Change in Sepsis in Mice Beáta Róka 1,Pál Tod 1,2, Tamás Kaucsár 1, Matej Vizovišek 3 , Robert Vidmar 3, Boris Turk 3,4 , Marko Fonovi´c 3,4,Gábor Szénási 1 and Péter Hamar 1,2,* 1 Institute of Translational Medicine, Semmelweis University, 1094 Budapest, Hungary; [email protected] (B.R.); [email protected] (P.T.); [email protected] (T.K.); [email protected] (G.S.) 2 Institute for Translational Medicine, Medical School, University of Pécs, 7624 Pécs, Hungary 3 Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, 1000 Ljubljana, Slovenia; [email protected] (M.V.); [email protected] (R.V.); [email protected] (B.T.); [email protected] (M.F.) 4 Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, 1000 Ljubljana, Slovenia * Correspondence: [email protected]; Tel.: +36-20-825-9751; Fax: +36-1-210-0100 Received: 18 November 2019; Accepted: 20 December 2019; Published: 27 December 2019 Abstract: (1) Background: Sepsis-induced acute kidney injury (AKI) is the most common form of acute kidney injury (AKI). We studied the temporal profile of the sepsis-induced renal proteome changes. (2) Methods: Male mice were injected intraperitoneally with bacterial lipopolysaccharide (LPS) or saline (control). Renal proteome was studied by LC-MS/MS (ProteomeXchange: PXD014664) at the early phase (EP, 1.5 and 6 h after 40 mg/kg LPS) and the late phase (LP, 24 and 48 h after 10 mg/kg LPS) of LPS-induced AKI. Renal mRNA expression of acute phase proteins (APP) was assessed by qPCR. (3) Results: Renal proteome change was milder in EP vs. LP. APPs dominated the proteome in LP (proteins upregulated at least 4-fold (APPs/all): EP, 1.5 h: 0/10, 6 h: 1/10; LP, 24 h: 22/47, 48 h: 17/44). Lipocalin-2, complement C3, fibrinogen, haptoglobin and hemopexin were the most upregulated APPs. Renal mRNA expression preceded the APP changes with peak effects at 24 h, and indicated renal production of the majority of APPs. (4) Conclusions: Gene expression analysis revealed local production of APPs that commenced a few hours post injection and peaked at 24 h. This is the first demonstration of a massive, complex and coordinated acute phase response of the kidney involving several proteins not identified previously. Keywords: acute kidney injury (AKI); lipopolysaccharide (LPS); renal acute phase reaction (APR); acute phase proteins (APP); mouse 1. Introduction Sepsis is estimated to be one of the leading causes of critical illness and mortality with constantly increasing incidence in intensive care units [1,2]. Sepsis is defined as an organ dysfunction of variable severities induced by a dysregulated immune response to an infection [3,4]. One of the organs determining early and late outcomes of sepsis is the kidney. Septic acute kidney injury (AKI) can be established within hours to days in up to 50% of intensive care unit patients [5]. In surviving patients, even mild or short episodes of AKI can predispose to an increased risk for developing chronic kidney disease and end-stage renal failure [4]. The acute phase response/reaction (APR) is part of the immune response to infections and tissue damage. Proteins whose plasma concentration is changed by at least 25% in response to Int. J. Mol. Sci. 2020, 21, 200; doi:10.3390/ijms21010200 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 20 The acute phase response/reaction (APR) is part of the immune response to infections and tissue Int. J. Mol. Sci. 2020, 21, 200 2 of 20 damage. Proteins whose plasma concentration is changed by at least 25% in response to pro- inflammatory stimuli are termed acute phase proteins (APP) [6–8]. They have a role in restoring pro-inflammatoryhomeostasis after inflammation stimuli are termed [7]. Although acute phase APPs proteins are thought (APP) to [ 6be–8 primarily]. They have produced a role in by restoring the liver homeostasisand secreted after into inflammationthe blood, they [7 ].are Although also synthesi APPszed are in thought other organs. to be primarily Thus, APPs produced can contribute by the liver to andlocal secreted defense intoresponses the blood, and theyrepair are mechanisms also synthesized [8]. in other organs. Thus, APPs can contribute to localPlasma defense responsesconcentrations and repairof APPs mechanisms (e.g., C-reactive [8]. protein) are well-established markers for diagnosingPlasma and concentrations monitoring sepsis of APPs [9]. (e.g.,Lipocalin-2 C-reactive (Lcn-2) protein) also termed are well-establishedas neutrophil gelatinase- markers forassociated diagnosing lipocalin and (NGAL) monitoring is a sensitive sepsis marker [9]. Lipocalin-2 of renal tubular (Lcn-2) injury also [10–13]. termed However, as neutrophil none of gelatinase-associatedthese biomarkers is lipocalin specific (NGAL) for sepsis is a sensitiveor AKI marker[9,14]. ofFor renal example, tubular injuryelevated [10– 13serum]. However, Lcn-2 noneconcentrations of these biomarkers were observed is specific in other for sepsispathologic or AKI conditions, [9,14]. For such example, as cardiovascular elevated serum [15] Lcn-2 and concentrationsgastrointestinal were[16,17] observed diseases. inSeveral other other pathologic AKI biomarker conditions, candidates such as have cardiovascular been identified [15 [14,18]] and gastrointestinalbut so far none of [16 them,17] diseases. was able Severalto differentiate other AKI the biomarker various types candidates of AKI havewith beensatisfactory identified specificity [14,18] butand so sensitivity far none [14,19]. of them For was this able reason, to diff thereerentiate is an the intensive various effort types to of identify AKI with biomarkers satisfactory or biomarker specificity andcombinations sensitivity that [14, 19could]. For be this used reason, for the there early is detection an intensive or follow-up effort to identify of AKI biomarkers [14]. or biomarker combinationsThe inflammatory that could beand used circulatory for the early effects detection of sepsis or follow-up are often of AKI modelled [14]. in rodents by administrationThe inflammatory of bacterial and circulatory wall endotoxin effects of sepsis(lipopolysaccharide: are often modelled LPS) in rodents[20,21]. byIncreased administration renal ofproduction bacterial wallof some endotoxin APPs, (lipopolysaccharide:such as fibrinogen [2 LPS)2], ceruloplasmin [20,21]. Increased [23], renal complement production C3 of[24,25], some APPs,haptoglobin such as[26–28], fibrinogen hemopexin [22], ceruloplasmin[29], serum amyloid [23], complement A [23,27], beta-2-microglobulin C3 [24,25], haptoglobin [22], α [26-1-acid–28], hemopexinglycoprotein [29 [23],], serum and plasminogen amyloid A [ 23activator,27], beta-2-microglobulin inhibitor-1 [30] have [22 been], α -1-aciddescribed glycoprotein in this model, [23], while and plasminogenrenal gene expression activator inhibitor-1of serum al [bumin30] have (a been negative described APP) inwas this shown model, to while be reduced renal gene [31]. expression Although ofthese serum scarce albumin and (aisolated negative findings APP) was demonstrate shown to bethe reduced renal production [31]. Although of thesesome scarceAPPs andin sepsis, isolated a findingscoordinated, demonstrate complex the APR renal of the production kidney has of some not been APPs evaluated in sepsis, before. a coordinated, complex APR of the kidneyIn hasthe notpresent been evaluatedstudy, we before.performed proteomic analysis to find relevant groups of proteins activatedIn the by present LPS study,at early we performed(1.5 h, 6 h) proteomic and late analysis (24 h, to and find 48 relevant h) time groups points of proteins after endotoxin activated byadministration LPS at early (1.5in the h, 6kidney. h) and Surprisingly, late (24 h, and we 48 fo h)und time abundant points after representation endotoxin administration of APPs among in thethe kidney.most regulated Surprisingly, proteins. we foundFurthermore, abundant qPCR representation measurements of APPs revealed among that the 14 most out of regulated these APPs proteins. were Furthermore,produced locally, qPCR in measurementsthe injured kidney. revealed that 14 out of these APPs were produced locally, in the injured kidney. 2. Results 2. Results 2.1. LPS-Induced Severe Inflammation in the Kidney 2.1. LPS-Induced Severe Inflammation in the Kidney All animals survived in the EP groups (40 mg/kg LPS), but one mouse died both in the LP24 and LP48All groups animals (10 survivedmg/kg LPS) in the during EP groups the experiment. (40 mg/kg LPS), but one mouse died both in the LP24 and LP48Endotoxin groups (10 significantly mg/kg LPS) duringupregulated the experiment. renal TNF-α and IL-6 mRNA production at all time points α (FigureEndotoxin 1). Both significantlyLPS doses caused upregulated severe, maximal renal TNF- inflammatoryand IL-6 mRNA response production in the kidneys. at all timeIL-6 mRNA points (Figurebut not1 TNF-). Bothα LPSmRNA doses started caused to decrease severe, maximal at 48 h. inflammatory response in the kidneys. IL-6 mRNA but not TNF-α mRNA started to decrease at 48 h. FigureFigure 1.1. Fold changes of TNF-α and IL-6 mRNA relative to the saline-treated control kidneyskidneys inin micemice afterafter lipopolysaccharidelipopolysaccharide (LPS)(LPS) administration.administration. ***: p ≤ 0.001. ( (aa)) early early phase phase (EP)1.5 (EP)1.5 and and EP6 groups. ≤ ((bb)) LP24LP24 andand LP48LP48 groups.groups. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 20 Int. J. Mol. Sci. 2020, 21, 200 3 of 20 Int.2.2. J. LPS-InducedMol. Sci. 2019, 20 Tubular, x FOR PEERDamage REVIEW in the Kidney 3 of 20 LPS significantly upregulated renal Lcn-2 mRNA and protein expression already from 1.5 h and 2.2. LPS-Induced Tubular Damage in the Kidney 2.2.from LPS-Induced 6 h, respectively Tubular (Figure Damage 2), in indicating the Kidney tubular injury.