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Subclinical Sequelae of acute Kidney InjuryPredispose To Acute Kidney Injury In Rats:Diagnostic BiomarkersIsabel Fuentes-Calvo 

University of Salamanca: Universidad de SalamancaCristina Cuesta 

University of Salamanca: Universidad de SalamancaSandra M. Sancho-Martinez 

University of Salamanca: Universidad de SalamancaOmar A. Hidalgo-Thomas 

Institute of Biomedical Research of SalamancaMaria Paniagua-Sancho 

Institute of Biomedical Research of SalamancaFrancisco J. Lopez-Hernandez 

Institute of Biomedical Research of SalamancaCarlos Martinez-Salgado  ( carlosms@usal.es )

University of Salamanca: Universidad de Salamanca https://orcid.org/0000-0003-4641-6717

Research

Keywords: acute kidney injury, diagnostic, predisposition, subclinical damage, urinary biomarkers

Posted Date: May 5th, 2021

DOI: https://doi.org/10.21203/rs.3.rs-449994/v1

License: This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License

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AbstractBackground. Acute kidney injury (AKI) is a risk factor for new AKI episodes and progression to chronickidney disease, cardiovascular events and death, as under certain circumstances renal repair ismaladaptive leading to proin�ammatory and pro�brotic signals. The de�nition for AKI recovery is basedon increases in plasma creatinine, but it only happens when 50-70 % of nephrons have been lost. Thereare no studies monitoring the real state of structural and functional recovery after an AKI episode. Weanalysed the functional and structural sequelae after an episode of AKI in rats, whether and for how longanimals retain enhanced sensibility to AKI and whether such a condition may be detected by knownbiomarkers of subclinical renal damage.

Methods. We performed a cisplatin (5mg/kg body weight)-induced AKI in rats and, after recovery, westudied the renal susceptibility to new AKI episodes after treatment with subtoxic doses of gentamicin (50mg/kg).

Results. One week after the recuperation of a cisplatin-induced AKI, the kidneys show histologicalalterations (cast formation, necrosis and tubular dilatation), and predisposition to new AKI episodes aftersubtoxic gentamicin treatment. Tubular function (urine output and urinary excretion of K+, assessed bythe furosemide tubular function test) remains affected despite the recovery of renal �ltration after the AKIepisode. Several urinary biomarkers detect this subclinical damage: albumin, transferrin, insulin-likegrowth factor-binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2). Theincreased [TIMP-2]*[IGFBP7] urinary excretion predicts new AKI episodes before gentamicin subtoxictreatment.

Conclusions. After a cisplatin-induced AKI, the kidneys are in functional risk for new AKI episodes, whichcan be prevented by monitoring IGFBP7 and TIMP-2 urinary excretion. Our �ndings require subsequentclinical validation to verify the potential of these biomarkers in predicting future episodes of kidneydamage.

BackgroundThe acute kidney injury (AKI) syndrome is characterized by an abrupt decline in renal function during aperiod of seven days or less, which is associated with high morbidity and mortality rates. [1, 2] Theincidence of AKI has increased in the last decade along with factors such as population aging andassociated diseases (e.g. diabetes and hypertension) and due to the use of stricter de�nitions of AKI. It isestimated that Aki occurs in 20–200 people per million inhabitants and affects 7–18 % of hospitalizedpatients, a percentage that increases up to 50 % in patients admitted to intensive care units. [2–4] AKI hastraditionally been considered a mostly reversible event with no consequences for long-term renalfunction. However, epidemiological studies have recently shown that AKI is a risk factor for the eventualloss of kidney function, for the progression to chronic kidney disease (CKD), and for other adverseoutcomes such as cardiovascular events and death. [2, 5–7]

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AKI may be preferentially caused by hemodynamic deregulation with no involvement of renal structures(i.e. pre-renal AKI), or by damage to renal tissues (i.e. intrinsic AKI). A most common form of intrinsic AKIis acute tubular necrosis, an affection of the tubular compartment often involving epithelial cell death.Although kidneys usually undergo complete functional recovery after an AKI episode due to the effectiveregenerative capacity of tubular cells, under undetermined circumstances renal repair becomesmaladaptive leading to proin�ammatory and pro�brotic signals, which eventually evolve to CKD. [8, 9]Accordingly, monitoring the repair process by means of non-invasive technology is an unmet butnecessary need to better handle AKI with precision medicine criteria.

An ideal de�nition of repair should be based on both restoration of kidney structure and recovery of renalfunction. A consensus de�nition of AKI recovery exists, which is based on the normalization (or return tobasal levels) of plasma creatinine (pCr). [3, 10] However, this de�nition carries the inherent limitations ofpCr. PCr increases over the normality range only when the function corresponding to at least 50–70 % ofnephrons has been lost. Consequently, after an AKI episode pCr may return to basal levels in the presenceof signi�cant residual structural alterations. [3, 11–13] The limitations of pCr to assess renal recovery arereinforced by epidemiological data showing an association between AKI and progression to CKD evenwhen pCr has returned to basal levels. [14, 15] Thus, it is critical to develop complementary methods todetect subclinical (i.e. under normal or basal pCr) renal alterations after an AKI episode.

During the last two decades, new biomarkers have been identi�ed that detect kidney damageindependently of pCr, even in subclinical circumstances. A few studies have explored the use of thesebiomarkers [i.e. insulin-like growth factor binding protein 7 (IGFBP-7), kidney injury molecule-1 (KIM-1),neutrophil gelatinase-associated lipocalin (NGAL), tissue metalloproteinase 2 (TIMP-2), interleukin 18,liver-type fatty acid-binding protein, N-acetyl-β-d-glucosaminidase], to monitor the recovery process. [9][12] Still, structural and functional recovery in the days and weeks immediately following the AKI episodehas not yet been su�ciently characterized. Post-AKI monitoring is necessary because AKI can evolve toAcute Kidney Disease (AKD), a condition in which AKI stage 1 or higher remains ≥ 7 days after an AKIevent. Of special interest is the stage 0A of AKD in which pCr has returned to basal levels and there is noevidence of injury or functional loss, but patients still retain a risk of further kidney damage. [2] AKDprovides an opportunity window to prevent future renal adverse outcomes [2, 16] and, therefore, newdiagnostic technology is needed to identify patients in early AKD.

The objective of this study was to characterize the evolution of renal repair and potential functional andstructural sequelae after pCr had returned to baseline levels following an episode of AKI in rats. Inparticular, we aimed at evaluating whether and for how long animals retain enhanced sensibility to AKI(i.e. higher risk of AKI) after pCr recovery and, if so, whether such a condition may be detected by knownbiomarkers of subclinical renal damage.

MethodsAll reagents were purchased from Merck (Madrid, Spain), except where otherwise indicated.

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In vivo experimental model

Male Wistar rats (240-260 g) were used in accordance with the Principles of the Declaration of Helsinkiand the European Guide for the Care and Use of Laboratory Animals (Directive 2010/63/UE) and Spanishnational and regional regulations (Law 32/2007/Spain, RD 1201/2005 and RD 53/2013). The BioethicsCommittee approved all procedures for Animal Care and Use of the University of Salamanca. Animalswere maintained under controlled environmental conditions, fed on standard chow and allowed to drinkwater ad libitum.

- Animal model 1: Evaluation of subclinical damage after AKI. Rats were subdivided into two groups (n=6per group and time point): control group (C): rats receiving saline solution (0.9% NaCl, i.p.); cisplatin group(CP): rats receiving cisplatin (5 mg/kg, i.p.), as in our previous studies. [17] Renal function was evaluatedin the following days: Basal (B), immediately prior to cisplatin administration; day of maximum kidneydamage (D4; i.e. the day of highest pCr); day of recovery (R0; i.e. the day in which pCr returns to basallevels); and one and two weeks after R0 (R1 and R2 respectively) (Figure 1).

- Animal model 2: Evaluation of predisposition to new AKI episodes. This experimental model wasperformed to assess if rats were predisposed to suffer new AKI episode when pCr had already returned tobasal levels after a previous AKI episode. We used our previously published model, in whichpredisposition to AKI is unmasked by treating rats with a sub-nephrotoxic dosage regime of gentamicin.[18] [19] [20] In this model, only predisposed rats (and not controls) undergo AKI when subject to sub-nephrotoxic gentamicin.

Rats were subdivided into 4 groups (n=6 each): cisplatin + gentamicin R0 group (R0 + G6): rats receivingcisplatin (5 mg/kg, i.p.) + gentamicin (50 mg/kg/day for 6 days, i.p., starting the day of recovery, R0);cisplatin + gentamicin R1 group (R1 + G6): rats receiving cisplatin (5 mg/kg, i.p.) + gentamicin (50mg/kg/day for 6 days, i.p., starting 1 week after recovery, R1); cisplatin + gentamicin R2 group (R2 + G6):rats receiving cisplatin (5 mg/kg, i.p.) + gentamicin (50 mg/kg/day for 6 days, i.p., starting two weeksafter recovery, R2); gentamicin control group (CT G6): rats receiving saline solution (i.p.) + gentamicin (50mg/kg/day, for 6 days, i.p., starting in R0, R1 and R2) (Figure 1)

Sample collection

At selected time points, urine and plasma samples were collected to evaluate renal function. For urinecollection, rats were individually allocated in metabolic cages. 24-hour urine was collected, centrifugedand stored at -80°C. Blood was drawn from the tail vein, centrifuged and plasma was stored at -80°C.Immediately before sacri�ce, kidneys were perfused by the aorta with saline solution and dissected. Onehalf was frozen in liquid nitrogen and subsequently kept at -80°C. The other half was �xed in buffered3.7% p-formaldehyde for histological studies.

Renal function studies

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Plasma (pCr) and urinary creatinine (uCr) and proteinuria were determined with commercial kits basedrespectively on the Jaffe reaction and the Bradford method, following the manufacturer´s instructions(BioAssay System, Hayward, CA USA). Glomerular �ltration rate (GFR) was estimated by the creatinineclearance (ClCr), using the formula: ClCr=uCr x UF/pCr, were UF stands for urine �ow.

The furosemide stress test (FST) was used to evaluate tubular integrity, as previously described. [21]Brie�y, a single dose of furosemide (20 mg/kg, i.p.) was administered to rats, and they were immediatelyallocated in metabolic cages to collect individual urine during the following hour. Urine volume and K+

excretion (LAQUAtwin B-731 Compact K+meter, Horiba Scienti�c, Kyoto, Japan) were measured asindicators of tubular performance. Rats with damaged tubuli show lower furosemide-induced urinaryvolume and K+ excretion, compared to control rats. [21]

Histological studies

Paraformaldehyde-�xed kidney samples were immersed in para�n, cut into 5 μm-thick slices and stainedwith haematoxylin and eosin (HE) and with Periodic acid–Schiff (PAS). Whole-kidney images wereobtained with photographs taken using the DotSlide virtual microscopy technique (Olympus BX51,Olympus Iberia, Barcelona, Spain). Images were analysed with the Olyvia Software (Olympus Iberia).

Tissue damage was quanti�ed using a severity score, as previously described. [22] Brie�y, 10 externalmedullar �elds [i.e. the area damaged by cisplatin [17, 23] were chosen from dot slide images of each rat,and each �eld was divided into 6 sections. In each section, four parameters were evaluated: necrosis,tubular dilatation or atrophy, hyaline deposits and epithelial disorganization. A score of 0–3 wasassigned to each parameter in a blind manner, according to the following criteria: 0, normal histology; 1,alteration in up to one-third of the section; 2, alteration from one-third to two-thirds of the section; 3,alteration in the whole area of the section. Section scores were added to give a �eld score (maximal scoreper �eld=12). The average score of 10 �elds was used for each kidney specimen.

Measurement of urinary biomarkers

ELISA kits were used to measure urinary levels of KIM-1 (Cusabio, Wuhan, China), NGAL (Bioporto,Hellerup, Denmark), albumin (Abcam), transferrin (Abcam, Cambridge, UK), TIMP-2 (Abcam) and IGFBP-7(Cloud Clone, Houston TX, USA), following the manufacturer instructions. The product of [TIMP-2]*[IGFBP7] urinary concentrations is marketed as NephroCheck®, and improves the diagnostic accuracyand outcomes of AKI patients. We have chosen these biomarkers due to previous studies by our researchgroup [19] and others [24] [25] which show their increased urinary levels in AKI patients and in differentexperimental models of AKI.

Statistical analysis

Statistical analysis was performed using the GraphPad Prism 7 software (San Diego, CA USA). Datanormal distribution was evaluated with the Shapiro-Wilk normality test. Data are shown as mean ±

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standard error of the mean (SEM) and dot blots. One-way ANOVA with Bonferroni´s test (for data withnormal distribution) or Dunn´s test (for data with non-normal distribution) were performed to compareexperimental groups; Spearman correlations were used to analyse the ability of biomarkers to predictrenal damage. P < 0.05 was considered statistically signi�cant.

ResultsThe kidneys show persisting structural alterations after functional recovery from AKI

Cisplatin treatment (5 mg/kg, i.p.) induces signi�cant increases in plasma creatinine levels (Figure 2A),proteinuria (Figure 2B) and a decrease in creatinine clearance (Figure 2C) after 4 days (D4); theseparameters return to baseline values about 4-6 days after D4 (R0). By D4, cisplatin treatment inducessevere structural alterations in the kidneys that include cast formation, tubular necrosis and tubulardilatation, as observed by HE staining (Figure 3B). In kidneys from rats with normalized renal function(i.e. at R0, R1 and R2), when plasma creatinine, proteinuria and creatinine clearance have returned tobasal levels, structural alterations, mainly tubular dilatation, are still present, although to a lower andtapering degree (Figure 3C-E). After quanti�cation of tissue damage, we noticed that these structuralalterations remain up to 2 weeks (i.e. R2) after recovery of renal function (Figure 3F). PAS staining showsthat the tubular epithelium brush border membrane is completely disorganized or absent in D4, and thatsituation does not improve signi�cantly when renal function parameters are normalized by R0 (Suppl.Figure 3B-C). The brush border membrane shows mild regeneration at R1 and R2, although signi�cantdamage remains, as compared with the normal structure observed in basal conditions or in controlanimals (Figure 4 D-E). Summarizing, the histological analysis shows that there are remarkable structuralalterations in the renal tissue after an episode of cisplatin-induced AKI, which persist when the renalfunction parameters return to baseline values.

Rats retain increased susceptibility to AKI after plasma creatinine normalization.

In rats that have undergone a previous cisplatin-induced AKI, treatment with the aminoglycosidegentamicin in subtoxic doses (50 mg/kg / day for 6 days, i.p.) promotes signi�cant new increases inplasma creatinine levels, proteinuria and a decrease in creatinine clearance when gentamicin isadministered the day of recovery (i.e. at R0) or one week after the day of recovery (i.e. at R1). However,this increase is not observed when subtoxic treatment is started two weeks after the recovery of cisplatin-induced AKI (i.e. at R2), nor in animals that have not had a previous AKI (control group) (Figure 5). Thesedata suggest that after a cisplatin-induced AKI episode, the kidneys remain in a state of increasedsusceptibility or predisposition to suffer a new AKI.

Alterations in the furosemide stress test parallel post-AKI predisposition to AKI

For its sensitivity to detect minimal and subclinical tubular alterations, [21] we applied the FST on AKIrats during the AKI episode and after pCr normalisation. Figure 6 shows that a profoundly reducedresponse to the FST (decreased urine output and urinary K+ excretion) is produced by cisplatin during AKI.

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This alteration progressively tapers off after AKI, but FST remains signi�cantly reduced after pCrnormalisation and up to two weeks thereafter, coinciding with the timing of the predisposition to AKI.

Urinary biomarkers detect subclinical damage after functional recovery from AKI, and correlates with newAKI episodes before subtoxic treatment with gentamicin

Cisplatin treatment promotes the urinary excretion of signi�cant levels of albumin, transferrin, IGFBP7,TIMP-2 and KIM-1. The highest urinary excretion of these biomarkers is mostly detected in D4, except forKIM-1 that peaks at R0. Although progressively declining, all these markers are still present at signi�cantlevels in urine when plasma creatinine is normalized (R0), and even two weeks later (Figure 7).

After recovering from a �rst AKI episode, the urinary excretion of transferrin, IGFBP7, TIMP-2 andIGFBP7*TIMP-2 is signi�cantly higher in rats predisposed to suffer new AKI episodes than in those whodo not present this predisposition (R2) (Figure 8B-E). Furthermore, there are highly signi�cant correlationsbetween the urinary levels of these markers in R0 and R1 (i.e. at the predisposition stage) and themagnitude of the subsequent decline in renal function produced after treatment with subtoxic doses ofgentamicin (i.e. increment in pCr and reduction in ClCr seen at R0+G6 and R1+G6) (Figure 8G).

DiscussionOur study shows that after the apparent clinical recovery from an AKI episode (that is, when pCr and ClCrhave returned to baseline levels), the kidney’s structure remains affected, at least for two weeks. Theselingering structural alterations coincide with an increased subclinical predisposition to new renal damage,and with an altered FST response and an increased urinary excretion of tubular injury biomarkers,including transferrin, IGFBP7 and TIMP-2.

The pathophysiological pro�ling and diagnosis of the condition known as predisposition to suffer AKI isa research �eld of clinical relevance. Several scoring models have been developed in the last 15 years,which predict the need of dialysis following surgical procedures with areas under the receiver operatingcharacteristic curve (ROC) of about 0.8, based on demographic and clinical variables. [26] [27] However,additional tools are needed to predict milder AKI not requiring dialysis, which is frequently involved in avariety of in-hospital outcomes. We have previously identi�ed a pCr-negative, urinary biomarker-negative,AKI-predisposing condition induced by sub nephrotoxic regimes of several drugs and toxins, includingcisplatin. [21] [18] [19] [28] In fact, these drug regimens cause absolutely no evidence of renal injury perse. However, there are no studies assessing the functional risk or predisposition to suffer a new AKI boutafter the apparent recovery from a previous episode. Our results indicate that, whilst basal renal functionis apparently normal, a subclinical, post-AKI predisposition to new AKI remains beyond pCr normalization,coinciding with incomplete structural recovery and with the presence of injury biomarkers in the urine.Monitoring repair by means of a non-invasive liquid biopsy may have high clinical repercussion, becauserepeated AKI is now known to be tightly associated with progression to AKD and CKD. [29] [2] [30] [31]This is stressed by the fact that the gold standard biomarker of AKI (i.e. pCr) is not suitable to monitorrepair, as its normalization does not preclude structural and functional sequelae. [32] It is thus of great

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clinical interest to pre-emptively identify individuals at increased risk of further AKI (even of subclinicalAKI), for optimal, preventive and bespoke clinical handling. Accordingly, there is thus an urgent need ofidentifying non-invasive biomarkers of predisposition to new kidney damage.

Interestingly, we found several subclinical markers that correlate not only with structural sequelae, butalso with the extant predisposition to AKI associated with them. A �rst marker is the response to the FST,which is altered in predisposed rats after pCr normalization. The loop diuretic furosemide inhibits theluminal Na-K-Cl cotransporter in the thick ascending limb of the loop of Henle and binds to the chloridetransport channel, and causes sodium, chloride, and potassium loss in urine, and osmotic diuresis.Previously, our research group has shown that the FST identi�es subclinical renal alterations associatedwith predisposition to AKI in the proximal tubule and thick ascending limb. In that study, predisposedanimals by a sub-nephrotoxic dose of cisplatin (i.e. no damage takes place) showed reduced furosemide-induced diuresis and K+ excretion. [21] In the present study, we observed that, during two weeks afterovert damage has occurred, the urine output and K+ urinary excretion forced by furosemide are alsosigni�cantly reduced, thus denoting that there are still alterations in tubular function that mostly affect itsresponse capacity (i.e. the tubular functional reserve). In agreement with this, as we showed previously,[21] the FST is a very sensitive but low-resolution tubule injury test. Accordingly, the FST is very useful fordetecting subtle alterations, but additional biomarkers are needed to inform damage granularity. In thissense, along with an altered FST, we also identi�ed increased urinary excretion of albumin, transferrin,IGFBP7 and TIMP-2 in connection with AKI sequelae and, some of them with the resulting predispositionto new AKI episodes.

These biomarkers are useful for the early and subclinical (i.e. with normal pCr) detection of AKI, and forthe estimation of AKI prognosis and evolution. Albuminuria is a known AKI biomarker, [33] independentlyassociated with a lower rate of AKI recovery in critically ill septic patients. [34] Increased albuminuria maybe the consequence of glomerular or tubular alterations. [35] [36] In our model, because of cisplatin’smechanisms of nephrotoxicity, [21] [37] albuminuria probably results from altered proximal tubularreclamation. The meaning of urinary transferrin reinforces this concept. In fact, our group has recentlyshown that urinary transferrin is a marker of tubular damage, and associates to the predisposition torenal damage induced by nephrotoxic insults causing subclinical tubular alterations, and not to othertypes of damage (i.e. vascular, haemodynamic). [20] The combination of [TIMP-2]*[IGFBP7], urinarymarkers of G1 cell cycle arrest, improve the identi�cation of patients at risk for imminent AKI, [38] [39] andthe decline in their urinary values is a valid predictor for renal recovery. [40] The exact mechanism bywhich IGFBP7 and TIMP-2 levels increase in the urine in different AKI models is not completely known,although increased �ltration, proximal tubular cell leakage and defects in tubular reabsorption are themost likely mechanisms. [24]

Our results also evidence the distinct biological role, and thus the potential diagnostic meaning, of eachof the so-called renal injury-biomarkers. Renal injury is an ambiguous term encompassing a variety ofalterations, each of which yielding speci�c markers. A challenge is thus to associate individualbiomarkers to concrete injury patterns, mechanisms or events, for a more speci�c, pathophysiological

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and personalized diagnosis of AKI. For instance, in our model the altered FST and the increased urinaryexcretion of transferrin, TIMP-1 and EGFBP7 all seem to portray a common pathological meaning, andthus indirectly link the observed post-AKI sequelae with the resulting predisposition to AKI. Conversely,urinary albumin and KIM-1 show some association with sequelae but not with predisposition, indicatingthat the undetermined (and probably different) pathological events producing their appearance in theurine in this model are not linked to the increased sensitivity to new AKI.

ConclusionsThis study shows that when plasma creatinine and creatinine clearance return to baseline values after anintrinsic AKI episode, both kidney structure and function may not be fully recovered. Our results suggestthat the follow-up of urinary transferrin, albumin and [TIMP-2]*[IGFBP7]), along with the FST, might beuseful for monitoring AKI sequelae and incomplete AKI repair. On the other hand, speci�cally transferrin,IGFBP7, TIMP-2, [TIMP-2]*[IGFBP7] and the FST also detect the increased predisposition to suffering anew episode of AKI, which may be used to pre-emptively identify patients at higher risk for appropriateand personalized handling.

DeclarationsEthics approval and consent to participate: Experimenal animals were used in accordance with thePrinciples of the Declaration of Helsinki and the European Guide for the Care and Use of LaboratoryAnimals (Directive 2010/63/UE) and Spanish national and regional regulations (Law 32/2007/Spain, RD1201/2005 and RD 53/2013). All procedures were approved by the Bioethics Committee for Animal Careand Use of the University of Salamanca.

Consent for publication: Does not apply

Availability of data and materials: Most data generated or analysed during this study are included in thispublished article. The complete datasets used and/or analysed during the current study are availablefrom the corresponding author on reasonable request.

Competing interests: The authors declare that they have no competing interests

Funding: This work was supported by grants from Instituto de Salud Carlos III, Ministerio de Ciencia eInnovación, Spain: PI15/01055, PI18/00996 and RETICS RD016/0009/0025 (REDINREN), co-funded byFEDER funds.

Authors' contributions:

Experimental work: IF-C, CC, SMS-M, OAH-T, MP-S

Data analysis: IF-C, CC

Study design, data interpretation, manuscript review and writing, funding acquisition: FJL-H, CM-S

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Acknowledgements: Cristina Cuesta and Omar A. Hidalgo-Thomas are recipients of two predoctoralfellowships, both funded by the Junta de Castilla y Leon (Spain) and the European Commission SocialFund

References1. Lameire N. The Pathophysiology of Acute Renal Failure. Crit Care Clin. 2005;21:197–210.

2. Chawla LS, Bellomo R, Bihorac A, Goldstein SL, Siew ED, Bagshaw SM, et al. Acute kidney diseaseand renal recovery: Consensus report of the Acute Disease Quality Initiative (ADQI) 16 Workgroup.Nat Rev Nephrol. 2017.

3. Kashani K, Kellum JA. Novel biomarkers indicating repair or progression after acute kidney injury.Curr Opin Nephrol Hypertens. 2015;24:21–7.

4. Negi S, Koreeda D, Kobayashi S, Yano T, Tatsuta K, Mima T, et al. Acute kidney injury: Epidemiology,outcomes, complications, and therapeutic strategies. Semin Dial. 2018.

5. Lv J, Zhang L. Renal Fibrosis: Mechanisms and Therapies. Springer Singapore; 2019.

�. Fortrie G, De Geus HRH, Betjes MGH. The aftermath of acute kidney injury: A narrative review of long-term mortality and renal function. Critical Care. 2019;23.

7. Rangaswamy D, Sud K. Acute kidney injury and disease: long-term consequences and management.Nephrology. 2018.

�. Lombardi D, Becherucci F, Romagnani P. How much can the tubule regenerate and who does it? Anopen question. Nephrol Dial Transplant. 2016;31:1243–50.

9. Forni LG, Darmon M, Ostermann M, Oudemans-van Straaten HM, Pettilä V, Prowle JR, et al. Renalrecovery after acute kidney injury. Intensive Care Med. 2017;43:855–66.

10. Bellomo R, Ronco C, Kellum JA, Mehta RL, Palevsky P, Acute Dialysis Quality Initiative workgroup.Acute renal failure - de�nition, outcome measures, animal models, �uid therapy and informationtechnology needs: the Second International Consensus Conference of the Acute Dialysis QualityInitiative (ADQI) Group. Crit Care. 2004;8:R204.

11. Göcze I, Wiesner C, Schlitt HJ, Bergler T. Renal recovery. Best Pract Res Clin Anaesthesiol.2017;31:403–14.

12. Goldstein SL, Chawla L, Ronco C, Kellum JA. Renal recovery. Crit Care. 2014;18:301.

13. Humphreys BD, Cantaluppi V, Portilla D, Singbartl K, Yang L, Rosner MH, et al. Targeting EndogenousRepair Pathways after AKI. J Am Soc Nephrol. 2016;27:990–8.

14. Heung M, Ste�ck DE, Zivin K, Gillespie BW, Banerjee T, Hsu CY, et al. Acute Kidney Injury RecoveryPattern and Subsequent Risk of CKD: An Analysis of Veterans Health Administration Data. In:American Journal of Kidney Diseases. W.B. Saunders; 2016. p. 742–52.

15. Bucaloiu ID, Kirchner HL, Norfolk ER, Hartle JE, Perkins RM. Increased risk of death and de novochronic kidney disease following reversible acute kidney injury. Kidney Int. 2012;81:477–85.

Page 11/21

1�. Kellum J a, Lameire N, Aspelin P, Barsoum RS, Burdmann E a, Goldstein SL, et al. KDIGO ClinicalPractice Guideline for Acute Kidney Injury. Kidney Int Suppl. 2012;2:1–138.

17. Ferreira L, Quiros Y, Sancho-Martínez SM, García-Sánchez O, Raposo C, López-Novoa JM, et al.Urinary levels of regenerating islet-derived protein III β and gelsolin differentiate gentamicin fromcisplatin-induced acute kidney injury in rats. Kidney Int. 2011;79:518–28. doi:10.1038/ki.2010.439.

1�. Vicente-Vicente L, Sánchez-Juanes F, García-Sánchez O, Blanco-Gozalo V, Pescador M, Sevilla MA, etal. Sub-nephrotoxic cisplatin sensitizes rats to acute renal failure and increases urinary excretion offumarylacetoacetase. Toxicol Lett. 2015;234:99–109.

19. Vicente-Vicente L, Ferreira L, González-Buitrago JM, López-Hernández FJ, López-Novoa JM, MoralesAI. Increased urinary excretion of albumin, hemopexin, transferrin and VDBP correlates with chronicsensitization to gentamicin nephrotoxicity in rats. Toxicology. 2013;304:83–91.

20. Casanova AG, Vicente-Vicente L, Hernández-Sánchez MT, Prieto M, Rihuete MI, Ramis LM, et al.Urinary transferrin pre-emptively identi�es the risk of renal damage posed by subclinical tubularalterations. Biomed Pharmacother. 2020;121:109684.

21. Casanova AG, Fuentes-Calvo I, Hernández-Sánchez MT, Quintero M, Toral P, Caballero MT, et al. Thefurosemide stress test and computational modeling identify renal damage sites associated withpredisposition to acute kidney injury in rats. Transl Res. 2020.

22. Quiros Y, Sánchez-gonzález PD, López-herńandez FJ, Morales AI, López-novoa JM. Cardiotrophin-1administration prevents the renal toxicity of iodinated contrast media in rats. Toxicol Sci.2013;132:493–501.

23. Sancho-Martínez SM, Blanco-Gozalo V, Quiros Y, Prieto-García L, Montero-Gómez MJ, Docherty NG, etal. Impaired Tubular Reabsorption Is the Main Mechanism Explaining Increases in Urinary NGALExcretion Following Acute Kidney Injury in Rats. Toxicol Sci. 2020;175:75–86.

24. Johnson ACM, Zager RA. Mechanisms underlying increased TIMP2 and IGFBP7 urinary excretion inexperimental AKI. J Am Soc Nephrol. 2018;29:2157–67.

25. Fan W, Ankawi G, Zhang J, Digvijay K, Giavarina D, Yin Y, et al. Current understanding and futuredirections in the application of TIMP-2 and IGFBP7 in AKI clinical practice. Clinical Chemistry andLaboratory Medicine. 2019;57:567–76.

2�. Pozzoli S, Simonini M, Manunta P. Predicting acute kidney injury: current status and futurechallenges. Journal of Nephrology. 2018;31:209–23.

27. Oh DJ. A long journey for acute kidney injury biomarkers. Renal Failure. 2020;42:154–65.

2�. Quiros Y, Ferreira L, Sancho-Martínez SM, González-Buitrago JM, López-Novoa JM, López-HernándezFJ. Sub-nephrotoxic doses of gentamicin predispose animals to developing acute kidney injury andto excrete ganglioside M2 activator protein. Kidney Int. 2010;78:1006–15.

29. Palant CE, Amdur RL, Chawla LS. The acute kidney injury to chronic kidney disease transition: Apotential opportunity to improve care in acute kidney injury. Contrib Nephrol. 2016;187:55–72.

30. Belayev LY, Palevsky PM. The link between acute kidney injury and chronic kidney disease. CurrentOpinion in Nephrology and Hypertension. 2014;23:149–54.

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31. Canaud G, Bonventre J V. Cell cycle arrest and the evolution of chronic kidney disease from acutekidney injury. Nephrology Dialysis Transplantation. 2015;30:575–83.

32. Murray PT, Mehta RL, Shaw A, Ronco C, Endre Z, Kellum JA, et al. Potential use of biomarkers inacute kidney injury: Report and summary of recommendations from the 10th Acute Dialysis QualityInitiative consensus conference. In: Kidney International. Nature Publishing Group; 2014. p. 513–21.

33. Dieterle F, Sistare F, Goodsaid F, Papaluca M, Ozer JS, Webb CP, et al. Renal biomarker quali�cationsubmission: A dialog between the FDA-EMEA and Predictive Safety Testing Consortium. NatureBiotechnology. 2010;28:455–62.

34. Neyra JA, Li X, Yessayan L, Adams-Huet B, Yee J, Toto RD. Dipstick albuminuria and acute kidneyinjury recovery in critically ill septic patients. Nephrology. 2016;21:512–8.

35. Tan HK, Lau YK. The signi�cance of tubular and glomerular proteinuria in critically ill patients withsevere acute kidney injury. Pakistan J Med Sci. 2014;30.

3�. Wijerathna TM, Mohamed F, Dissanayaka D, Gawarammana I, Palangasinghe C, Shihana F, et al.Albuminuria and other renal damage biomarkers detect acute kidney injury soon after acuteingestion of oxalic acid and potassium permanganate. Toxicol Lett. 2018;299:182–90.

37. Snchez-Gonzlez PD, López-Hernández FJ, López-Novoa JM, Morales AI. An integrative view of thepathophysiological events leading to cisplatin nephrotoxicity. Critical Reviews in Toxicology.2011;41:803–21.

3�. Kashani K, Al-Khafaji A, Ardiles T, Artigas A, Bagshaw SM, Bell M, et al. Discovery and validation ofcell cycle arrest biomarkers in human acute kidney injury. Crit Care. 2013;17.

39. Dewitte A, Joannès-Boyau O, Sidobre C, Fleureau C, Bats ML, Derache P, et al. Kinetic eGFR and novelAKI biomarkers to predict renal recovery. Clin J Am Soc Nephrol. 2015;10:1900–10.

40. Meersch M, Schmidt C, Van Aken H, Martens S, Rossaint J, Singbartl K, et al. Urinary TIMP-2 andIGFBP7 as early biomarkers of acute kidney injury and renal recovery following cardiac surgery. PLoSOne. 2014;9.

Figures

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Figure 1

Temporal diagram of the experimental models and collection of plasma, urine and kidney tissue samples.B: basal; CDDP: cisplatin treatment; CT: control; d4: day of maximum kidney damage after cisplatintreatment; R0: day of recovery; R0+G6: six days of gentamicin treatment after R0; R1: one week afterrecovery; R1+G6: six days of gentamicin treatment after R1; R2: two weeks after recovery; R2+G6: sixdays of gentamicin treatment after R2

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Figure 2

Renal function parameters: Plasma creatinine (PCr) (A), proteinuria (B) and creatinine clearance (CrCl)(C). B: basal; CDDP: cisplatin treatment (5 mg/kg body weight) group; CT: control group; D4: day ofmaximum kidney damage after cisplatin treatment; R0: day of recovery; R1: one week after recovery; R2:two weeks after recovery; R2+G6: six days of gentamicin treatment (50 mg/kg body weight) after R2. P<0.01: * vs B, # vs D4

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Figure 3

Renal histology: histological damage. Hematoxylin-eosin (H-E) staining in control group (A), D4 (B), R0(C), R1 (D) and R2 (E); quanti�cation of histological damage (score) (F). P< 0.01: * vs B, # vs D4, $ vs R0

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Figure 4

Renal histology: brush border membrane disorganization. Periodic Acid-Schiff (PAS) staining in controlgroup (A), D4 (B), R0 (C), R1 (D) and R2 (E).

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Figure 5

Susceptibility to AKI: Plasma creatinine (PCr) (A-C), proteinuria (D-F) and creatinine clearance (CrCl) (G-I).B: basal; CDDP: cisplatin treatment (5 mg/kg body weight) group; CT: control group; D4: day of maximumkidney damage after cisplatin treatment; G: gentamicin administration; R0: day of recovery; R0+G6: sixdays of gentamicin treatment (50 mg/kg body weight) after R0; R1: one week after recovery; R1+G6: sixdays of gentamicin treatment (50 mg/kg body weight) after R1; R2: two weeks after recovery; R2+G6: sixdays of gentamicin treatment (50 mg/kg body weight) after R2. P< 0.01: * vs B, # vs D4, $ vs R0, & vs R1

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Figure 6

Furosemide stress test. Plasma creatinine (PCr) (A), urine output (B) and urinary potassium excretion (C)after administration of furosemide (20 mg/kg, i.p.). B: basal; CDDP: cisplatin treatment (5 mg/kg) group;CT: control group; D4: day of maximum kidney damage after cisplatin treatment; R0: day of recovery; R1:one week after recovery; R2: two weeks after recovery. P< 0.01: * vs B, # vs D4, $ vs R0

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Figure 7

Urinary excretion of biomarkers. Albumin (A), transferrin (B), IGFBP7 (C), TIMP-2 (D) and KIM-1 (E) urinaryexcretion. B: basal; CDDP: cisplatin treatment (5 mg/kg body weight) group; CT: control group; D4: day ofmaximum kidney damage after cisplatin treatment; R0: day of recovery; R1: one week after recovery; R2:two weeks after recovery. P< 0.05: * vs B, # vs D4, $ vs R0; P< 0.01: ** vs B, # # vs D4, $$ vs R0

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Figure 8

Predictive value of urinary biomarkers. Albumin (A), transferrin (B), IGFBP7 (C), TIMP-2 (D), IGFBP7*TIMP-2 (E) and KIM-1 (F) urinary excretion in rats with and without predisposition to a new AKI episode, andstatistical correlations (G) between urinary levels of biomarkers in recuperation points (R0, R1, R2) withthe increase in plasma creatinine and the decrease in creatinine clearance determined after six days of

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subtoxic gentamicin treatment (50 mg/kg body weight) after every recuperation point (R0+G6, R1+G6,R2+G6).