TY - JOUR
AU - López-Hernández, Francisco, J
AB - Abstract Acute kidney injury (AKI) is a serious syndrome with increasing incidence and health consequences, and high mortality rate among critically ill patients. Acute kidney injury lacks a unified definition, has ambiguous semantic boundaries, and relies on defective diagnosis. This, in part, is due to the absence of biomarkers substratifying AKI patients into pathophysiological categories based on which prognosis can be assigned and clinical treatment differentiated. For instance, AKI involving acute tubular necrosis (ATN) is expected to have a worse prognosis than prerenal, purely hemodynamic AKI. However, no biomarker has been unambiguously associated with tubular cell death or is able to provide etiological distinction. We used a cell-based system to identify TCP1-eta in the culture medium as a noninvasive marker of damaged renal tubular cells. In rat models of AKI, TCP1-eta was increased in the urine co-relating with renal cortical tubule damage. When kidneys from ATN rats were perfused in situ with Krebs-dextran solution, a portion of the urinary TCP1-eta protein content excreted into urine disappeared, and another portion remained within the urine. These results indicated that TCP1-eta was secreted by tubule cells and was not fully reabsorbed by the damaged tubules, both effects contributing to the increased urinary excretion. Urinary TCP1-eta is found in many etiologically heterogeneous AKI patients, and is statistically higher in patients partially recovered from severe AKI. In conclusion, urinary TCP1-eta poses a potential, substratifying biomarker of renal cortical damage associated with bad prognosis. acute kidney injury, acute tubular necrosis, apoptosis, TCP1-eta, urinary biomarker, tubular injury, bad prognosis Acute kidney injury (AKI) is a syndrome of sudden renal excretory dysfunction with serious health consequences and high cost, consuming 1% of the total health budget and 5% of hospital expenditure (Kerr et al., 2014; Sancho-Martínez et al., 2015; Vandijck et al., 2007). Worldwide, the overall mortality rate of AKI is 23.9% in adults and 13.8% in children (Susantitaphong et al., 2013). In intensive and critical patients, the mortality rate may reach 50%–80%, especially when AKI occurs in the context of multiorgan failure (Block and Schoolwerth, 2006; Kellum and Hoste, 2008; Waikar et al., 2008). A fraction of the patients never recover previous renal function (Rachoin et al., 2012). For instance, 7.5% of patients require permanent dialysis after AKI, a number that grows to 40%–60% among those with prior chronic kidney disease (CKD) (González et al., 2008). Independently, AKI also increases the odds of progressing to CKD (Bucaloiu et al., 2012; Hsu, 2012). A worse outcome has been linked to AKI severity, with repeated episodes, and prior CKD (Belayev and Palevsky, 2014; Canaud and Bonventre, 2015). However, transient (Brown et al., 2012; Coca et al., 2010), mild (Praught and Shlipak, 2005), and even subclinical AKI (Albert et al., 2018; de Geus et al., 2017; Ronco et al., 2012) impact the outcome. The pathogenesis, etiology and AKI type are also associated with prognosis. In this sense, AKI has been traditionally classified into 3 types, namely prerenal, renal (or intrinsic), and postrenal (Clarkson et al., 2008; Kaufman et al., 1991; Uchino, 2010), with distinct etiopathology. Prerenal AKI (ie, prerenal azotemia) is considered to be the mildest form. In prerenal AKI, kidney structures are preserved and, consequently, it is associated with better clinical outcomes than intrinsic AKI, which involves renal parenchymal damage (Esson and Schrier, 2002; Kaufman et al., 1991; Lee et al., 2007; Liaño and Pascual, 1996; Rachoin et al., 2012; Uchino et al., 2012). The most common pattern of intrinsic AKI is acute tubular necrosis (ATN). Acute tubular necrosis is a rather ambiguous term comprising primary and heterogeneous damage to the renal tubular compartments (Endre et al., 2013; Sancho-Martínez et al., 2015), including sublethal alterations in tubule cells, compromising tubular function (Heyman et al., 2002; Rosen and Stillman, 2008). The prognosis of patients presenting mild, sublethal alterations may differ substantially from that of patients with extensive tissue destruction. Cataloging the whole range of symptoms between both extremes under a unique ATN concept abrogates effective classification and stratification criteria-based care, which is crucial for patient outcome. Hence, new diagnostic criteria and biomarkers are necessary to subclassify the concept of ATN into distinct pathophysiological scenarios (Sancho-Martínez et al., 2015). The etiological diagnosis of AKI and patient stratification are still poorly performed retrospectively, based on the duration of the episode and the response to fluid therapy (Nejat et al., 2012; Rachoin et al., 2012; Uchino, 2010; Uchino et al., 2012). In addition, an undetermined amount of prerenal AKI cases involve a variable degree of renal damage, as an initial stage of a complex continuum, which further complicates diagnosis (Nejat et al., 2012), and the immediate and prospective patient triage on solid rather than on exclusion criteria. Prospectively, a pathophysiological diagnosis based on more specific biomarkers will help to improve prognosis estimation (Endre et al., 2013), and enable precision medicine and personalized clinical handling. For this purpose, the gold standard biomarker [ie, plasma creatinine (Crpl)] conveys no etiological information as it increases in all forms of AKI (Endre et al., 2013; Sancho-Martínez et al., 2015). Biomarkers of subclinical AKI such as neutrophil gelatinase-associated lipocalin (NGAL) distinguish prerenal from intrinsic AKI (Nejat et al., 2012; Singer et al., 2011; Vanmassenhove et al., 2013), although this capacity is also questionable (Nejat et al., 2012). In fact, NGAL also increases in the urine as a consequence of systemic or extrarenal inflammation (Oikonomou et al., 2012), and in different chronic nephropathies (Endre et al., 2013; Tasanarong et al., 2013). In addition, none of these markers has shown differential diagnosis capacity among ATN patterns. Accordingly, more specific markers of differentiated pathophysiological scenarios, events, and mechanisms are needed. We studied the supernatant of tubular cells cultured with nephrotoxic drugs, to evaluate the released biomarkers which could eventually be found in the urine following tubular injury. We identified the tail-less complex polypeptide-1 eta subunit (TCP1-eta) in the supernatant of tubular cells undergoing apoptosis, and subsequently reported the presence in the urine of rats with ATN in correlation with the extent of renal cortical damage, and also in AKI patients, in correlation with bad prognosis (ie, incomplete recovery from AKI). Tail-less complex polypeptide-1 eta (TCP1-η) is part of the double-ring TCP-1 complex. Each ring is composed of 8 approximately 59 kDa subunits, α, β, γ, δ, ϵ, ζ, η, and θ. Tail-less complex polypeptide-1 is involved in the folding of scaffolding and cytoskeletal proteins, including actin and tubulin, and participates in cellular shape conformation and responses to detachment and stress (Liang et al., 2014). MATERIALS AND METHODS Reagents, cells, and protocols Unless otherwise indicated, reagents were purchased from Sigma (Madrid, Spain). A mouse cortical tubule cell line (MCT) was used for the study. Cells were maintained in RPMI 1640 medium (Gibco, Madrid, Spain) supplemented with 10% fetal bovine serum (FBS, Gibco), 500 U/ml penicillin, 50 mg/ml streptomycin (Gibco), and 1 mM l-glutamine. Cells were treated in the medium without serum for 9 and 18 h with cisplatin (0–100 μM), or for 18 h with cycloheximide (0–800 μM). During treatments, cells were photographed under a live cell, light microscope (Axiovert 200 M, Zeiss; and Nikon Eclipse TS100, Barcelona, Spain) for visual inspection. Finally, cell extracts were obtained in the extraction buffer (25 mM PIPES, pH 7; 25 mM KCl; 5 mM EGTA; 0.5% NP-40, and a mixture of protease inhibitors consisting of 1 mM phenylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml aprotinin). The protein content was determined using a commercial kit (Bio-Rad, Madrid, Spain) based on the Lowry method. MTT assay Viable cell numbers were comparatively estimated by incubating cell cultures with 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) for 4 h. Next, 10% SDS in 0.01 M HCL was added in a ratio of 1:1 (vol/vol) and incubated overnight at 37°C. Finally, absorbance was measured at 570 nm as an index of viable cell number. Caspase activity Twenty micrograms of protein from cell extracts was incubated with 100 μl reaction buffer (50 mM HEPES, pH 7.4; 100 mM NaCl; 1 mM EDTA; 0.1% 3-[3-(cholamidopropyl)-dimethylammonio]-1-propane sulfonate; 10% sucrose; and 5 mM DTT) containing 100 μM of the fluorogenic caspase substrate rhodamine 110, bis-(N-CBZ-l-aspartyl-l-glutamyl-l-valyl-l-aspartic acidamide) (Z-DEVD)2Rh110 (Anaspec, Fremont, California) at 37°C for 30 min in a Fluoroskan Ascent FL fluorometer (Thermo, Rockford, Illinois), and fluorescence intensity was measured every 3 min at 538 nm, upon excitation at 485 nm. Caspase activity was calculated from the slope of the linear relationship between fluorescence intensity and time. Western blot Briefly, 20 μl from each supernatant, 30 μg from each cell extract, a volume of urine from each rat corresponding to the same fraction of 24-h urinary output, and 21 μl per human urine sample were separated by acrylamide electrophoresis. Proteins were transferred to an Immobilon-P Transfer Membrane (Millipore, Madrid, Spain) and incubated with anti TCP1-eta antibody (NPB2-20588, Novus Biologicals, Littleton, Colorado), followed by horseradish peroxidase-conjugated secondary antibodies and chemiluminescent detection (Immobilon Western Chemiluminescent HRP Substrate kit; Millipore) with photographic films (Kodak, Madrid, Spain). DNA fragmentation DNA fragmentation was determined with the commercial kit Cell Death Detection Plus (Roche Diagnostics, Barcelona, Spain) using 10 μg of protein from each cell extract. Lactose dehydrogenase assay Lactose dehydrogenase (LDH) was measured in 50 μl culture supernatants with a CytoTox 96 nonradioactive cytotoxicity assay (Promega, Barcelona, Spain). RT-qPCR analysis Total RNA was isolated using Nucleospin RNAII (Macherey-Nagel, Düren, Germany). First-strand cDNA was generated from 1 μg of total RNA using poly-dT as a primer with the M-MLV reverse transcriptase (Promega). Real-time PCR (qPCR) was performed in triplicate. In a total reaction volume of 20 μl containing 1 μl of cDNA, 0.4 μM of each primer and 1× iQSybrGreen Supermix (Bio-Rad), with initial denaturation at 95°C for 5 min, followed by 40 cycles of 30 s at 95°C, and 30 s at 65°C, and finally, 1 cycle of 30 s at 72°C. Threshold cycle (Ct) values were obtained. The sequences of TCP1-eta used were: sense 5′-GCCGCTTCCAAGATGATGCCCA-3′ anti-sense 5′-CGGGGACCCAGGGTGGTTCT-3′; and ribosomal protein S13 (RPS13): sense 5′-GATGCTAAATTCCGCCTGAT-3′ anti-sense 5′-TAGAGCAGAGGCTGTGGATG-3′. Standard curves were run for each transcript to assure exponential amplification and to rule out nonspecific amplification. Changes in the expression level were calculated following normalization with RPS13. The reactions were run on an iQ5 Real-time PCR detection system (Bio-Rad). Proteomic studies The culture supernatant of MCT cells treated with cisplatin or vehicle (as controls) in serum free medium for 18 h was analyzed by 2-dimensional electrophoresis (2-DE), as previously described in Quiros et al. (2010) and Ferreira et al. (2011). In short, proteins (200 µg) were precipitated, isoelectrically focused (500–8000 V) through 18-cm-long immobilized pH gradient strips, pH 3-11NL (GE Healthcare, Madrid, Spain), further separated in 12% SDS-polyacrylamide gels, fixed and stained with Sypro Ruby (Molecular Probes, Barcelona, Spain). The spots of interest were analyzed with the Image Master Platinum software (GE Healthcare) and digested in-gel with porcine trypsin (Promega). Tryptic peptides were analyzed by MALDI-TOF on an Autoflex III instrument (Bruker Daltonics, Madrid, Spain). Protein identification was performed with the MASCOT software (www.matrixscience.com). In case of all protein identifications, the probability scores were greater than the score fixed by MASCOT as significant with a p value lower than .05. Rat models of AKI Male Wistar rats (220–240 g) were housed under controlled experimental conditions and allowed free access to regular chow and water. Rats were handled in accordance with the Declaration of Helsinki and Principles on the Advice on Care and Use of Animals referred to in: law 14/2007 (3 July) on Biomedical Research, Conseil de ĺEurope (published in Official Daily N. L358/1-358/6, December 18, 1986), Spanish Government (Royal Decree 53/2013 of February 1, published in Official Bulletin of the State 34, Sec. I, pages 11370–11421, February 8, 2013). Rats were divided into 6 groups (n = 5 per group): (1) control group (control), saline solution; (2) ischemia-reperfusion group (I/R), 1 h of warm renal ischemia plus contralateral nephrectomy; (3) cisplatin group (CISP), a single ip administration of 5 mg/kg cisplatin; (4) gentamicin group (GENTAM), gentamicin 150 mg/kg ip per day for 6 days; (5) prerenal group (PR), ibuprofen 400 mg/kg plus trandolapril 0.7 mg/kg po for 10 days, and from the 4th day furosemide 20 mg/kg ip per day for 6 days; (6) maleate group (Maleate, n = 4), a single iv injection of sodium maleate (400 mg/kg). Rats were introduced in metabolic cages to obtain 24-h, individual urine samples. Blood was collected from the tail vein, and plasma was obtained by centrifugation. Finally, at the end of the treatments, kidneys were perfused with heparinized saline and immediately dissected. Half of the kidney was fixed in paraformaldehyde for histological studies, and the rest was frozen at −80°C for biochemical analysis. Renal function Renal function was monitored by means of Crpl and urea and urinary protein concentration, which were determined using commercial colorimetric kits (Quantichrom Creatinine and Quantichrom Urea Assay Kits, BioAssay Systems, Hayward, California), in accordance with the manufacturer’s instructions. Renal histopathology Paraformaldehyde-fixed tissue samples were immersed in paraffin, sectioned into 5-µm-thick slices and stained with hematoxylin and eosin. Tissue slices were also immunostained with anti TCP1-eta antibody (Novus Biologicals) and anti-caspase-3 antibody (Cell Signaling, Danvers, Massachusetts); followed by horseradish peroxidase conjugated secondary antibodies. Renal injury was semiquantitated with a severity score, as described in Quiros et al. (2013). Briefly, 10 cortical fields and 10 corticomedullary fields were photographed under light microscopy (×400), and each field was divided into 10 sections. A score of 0–3 was assigned to each section in a blind manner, according to the following criteria: 0, normal histology; 1, tubular cell swelling, brush border loss, nuclear condensation, or nuclei loss in up to one-third of the section; 2, same as 1, but from one-third to two-thirds of the section; 3, same as 1, but in the whole area of the section. Section scores were added to give a field score (maximal score per field = 30). The average score of 10 fields was used for each kidney specimen. In situ renal perfusion Male Wistar rats (220–240 g) were divided into the following groups: (1) Control (n = 6): Saline solution; (2) Cisplatin (CISPL 5, n = 6): A single ip injection of 5 mg/kg cisplatin. At the end of the experimental protocol or treatment, rats were anesthetized and an extracorporeal circuit for kidney perfusion was set up, as previously described in Blázquez-Medela et al. (2014). Briefly, the renal artery, vein and ureter of the right kidney were ligated. The renal artery and vein of the left kidney and the urinary bladder were cannulated. Oxygenated and warm (37°C) Krebs-dextran was perfused through the renal artery at 3 ml/min. The effluent from the renal vein was collected. Urine fractions were collected from a catheter placed in the urinary bladder, starting prior to perfusion with Krebs (when blood was still circulating through the kidney), and during perfusion with Krebs for 60 min. As a control and to eliminate experimental artifacts, kidneys were also perfused in situ with blood from the carotid artery, and urine was collected as above. Patients Urine samples were collected from 86 volunteers from the Nephrology Department at the Hospital Universitario Marqués de Valdecilla (Santander, Spain): 68 patients presented prerenal or renal AKI; 6 were disease controls, and 12 healthy individuals. The hospital provided renal function and diagnosis data. Glomerular filtration rate was estimated using the Cockroft-Gault equation. Statistical analysis Cell and animaldata are represented as the mean ± SD or SEM of n experiments, as indicated in each case. Statistical comparisons were assessed using the 1-way ANOVA analysis, followed by the Bonferroni post hoc test. Patients’ data normality was assessed with the D'Agostino & Pearson normality test. Nonparametric data were compared using the Mann-Whitney U test (2-tailed). Box plot graphics display the median, quartiles, and ranges (10–90 percentiles). Correlation analyses were performed using the Spearman correlation tests. Logistic regression analysis was used to study the predictive capacity of TCP1-eta on patient recovery from AKI. GraphPad Prism 7.0 (GraphPad Software, San Diego, California) and the IBM SPSS Statistics 23 (IBM, Madrid, Spain) software were used to perform statistical analyses. A p value <.05 was considered statistically significant. RESULTS Cisplatin Induces Apoptosis of MCT Cells and a Parallel Shedding of TCP1-eta to the Culture Medium The cisplatin concentration-effect relationship was studied in MCT cells cultured in medium without FBS. Serum withdrawal was implemented to avoid interference of serum proteins with MCT-secreted proteins. Mouse cortical tubule cells responded to cisplatin in a similar way to the presence of serum with respect to cytotoxic potency and efficacy (comparative data not shown). After 18 h of treatment, cisplatin, in a concentration-dependent manner, reduced cell viability and increased the number of apoptotic cells (Figs. 1A–C), caspase (DEVDase) activity (Figure 1D) in the absence of plasmalemmal rupture (ie, no LDH release; Figure 1E), and internucleosomal DNA fragmentation (Figure 1F), all hallmarks of apoptosis (Núñez et al., 2010). Figure 1. Open in new tabDownload slide Antiproliferative effect and morphological phenotypes induced by cisplatin. A, MTT-based proliferation/viability, concentration-effect profile of mouse cortical tubule (MCT) treated for 18 h with cisplatin (0–30 μM). Data represent the average ± SD of n = 3. *p < .05 with respect to control. B, Quantification of the number of apoptotic cells after 18 h of treatment with cisplatin (0–30 μM). C, Representative light microscopy photographs (n = 3, ×400) of MCT cells treated with 0 (control), 10 and 30 μM cisplatin for 18 h. White arrows: apoptotic cells. D, DEVDase (caspase) activity in cell extracts, (E) lactose dehydrogenase activity in culture medium, (F) internucleosomal DNA fragmentation. Data represent the average ± SD of n = 3. *p < .05 with respect to 0 μM cisplatin in its group; #p < .05 with respect to the same concentration of cisplatin after 9 h of treatment. Figure 1. Open in new tabDownload slide Antiproliferative effect and morphological phenotypes induced by cisplatin. A, MTT-based proliferation/viability, concentration-effect profile of mouse cortical tubule (MCT) treated for 18 h with cisplatin (0–30 μM). Data represent the average ± SD of n = 3. *p < .05 with respect to control. B, Quantification of the number of apoptotic cells after 18 h of treatment with cisplatin (0–30 μM). C, Representative light microscopy photographs (n = 3, ×400) of MCT cells treated with 0 (control), 10 and 30 μM cisplatin for 18 h. White arrows: apoptotic cells. D, DEVDase (caspase) activity in cell extracts, (E) lactose dehydrogenase activity in culture medium, (F) internucleosomal DNA fragmentation. Data represent the average ± SD of n = 3. *p < .05 with respect to 0 μM cisplatin in its group; #p < .05 with respect to the same concentration of cisplatin after 9 h of treatment. The protein content in the culture medium of MCT cells treated for 18 h with 0–30 μM cisplatin was studied using 2D proteomic analysis. The secretomes were very similar in all conditions (Figure 2A). However, a specific protein-spot consistently increased in the culture medium after treatment with cisplatin (Figure 2A) and was unambiguously identified by mass spectrometry as TCP1-eta. Western blot analysis confirmed that the level of this protein increased with increasing concentrations of cisplatin (Figure 2B). RT-qPCR experiments demonstrated progressive TCP1-eta gene induction with increasing concentrations of the drug (Figure 2C, left panel), in close relation with secretion. However, the expression of the housekeeping gene (RPS13) was not modified (Figure 2C, right panel). Tail-less complex polypeptide-1 eta did not accumulate inside the cells, as revealed by Western blot analysis of cell extracts (Figure 2D), indicating that the excessive expression of this protein was evacuated by the cells. Other apoptosis-related proteins, such as t-gelsolin (a proteolytic fragment of gelsolin generated by caspases) were not detected in the culture medium (Figure 2E). Figure 2. Open in new tabDownload slide Differential proteomic profiling of the culture medium from control and cisplatin-treated cells. A, Representative images of 2-DE gels, n = 3. The marked spots correspond to proteins significantly increased in the culture medium of mouse cortical tubule (MCT) cells after treatment for 18 h with 3 or 30 μM cisplatin with respect to the control. B, Representative image of Western blot analysis of tail-less complex polypeptide-1 eta (TCP1-eta) in culture medium, after 9 and 18 h, in MCT cells treated with cisplatin (0–30 μM). Data represent the average ± SD of n = 3 independent experiments; **p < .01 with respect the control. C, Expression of TCP1-eta (left panel) and ribosomal protein S13 (right panel) mRNA in MCT cells treated with 0–30 μM cisplatin for 18 h (n = 3). Data are expressed as the average ± SD. **p < .01 versus control. D, Representative image of Western blot analysis of intracellular TCP1-eta and α-tubulin (as loading control). E, Representative image of Western blot analysis of t-gelsolin in the culture medium; in MCT cells treated with cisplatin (0–30 μM) for 9 and 18 h. Data represent the average ± SD of n = 2 independent experiments. Figure 2. Open in new tabDownload slide Differential proteomic profiling of the culture medium from control and cisplatin-treated cells. A, Representative images of 2-DE gels, n = 3. The marked spots correspond to proteins significantly increased in the culture medium of mouse cortical tubule (MCT) cells after treatment for 18 h with 3 or 30 μM cisplatin with respect to the control. B, Representative image of Western blot analysis of tail-less complex polypeptide-1 eta (TCP1-eta) in culture medium, after 9 and 18 h, in MCT cells treated with cisplatin (0–30 μM). Data represent the average ± SD of n = 3 independent experiments; **p < .01 with respect the control. C, Expression of TCP1-eta (left panel) and ribosomal protein S13 (right panel) mRNA in MCT cells treated with 0–30 μM cisplatin for 18 h (n = 3). Data are expressed as the average ± SD. **p < .01 versus control. D, Representative image of Western blot analysis of intracellular TCP1-eta and α-tubulin (as loading control). E, Representative image of Western blot analysis of t-gelsolin in the culture medium; in MCT cells treated with cisplatin (0–30 μM) for 9 and 18 h. Data represent the average ± SD of n = 2 independent experiments. Tail-less Complex Polypeptide-1 eta Is Also a Marker of Cycloheximide Cytotoxicity To study whether the increased TCP1-eta net secretion also occurred in case of other cytotoxic stimuli, MCT cells were also treated with cycloheximide (50–800 μM) or vehicle (as control) for 18 h. Cycloheximide (1) reduced cell viability (Figure 3A); (2) induced apoptosis, evidenced by the detection of cells with apoptotic phenotype (Figure 3B) and executioner caspase 3 activation (Figure 3C); and also (3) increased TCP1-eta secretion in the culture medium (Figure 3D). Figure 3. Open in new tabDownload slide Cycloheximide also induces an apoptotic phenotype and tail-less complex polypeptide-1 eta (TCP1-eta) release, in a concentration-dependent manner. Mouse cortical tubule cells treated with 0 (control), 50, 100, and 200 μM cycloheximide for 18 h. At the end of the experiment, (A) an MTT-based proliferation/viability was performed and (B) the death phenotype was determined by light microscopy, for the presence or absence of cell dismantling into apoptotic bodies (representative images are shown, ×400). The dotted line marks the starting point (time = 0 h). White arrows: apoptotic cells. C, Representative images of Western blot analysis after 18 h of treatment with cycloheximide of active caspase 3 (p19 fragment, n = 2), densitometry quantification, and representative images of Western blot analysis of α-tubulin (as loading control). D, TCP1-eta in the culture medium (n = 3), densitometry quantification, and representative images of Western blot analysis of α-tubulin (as loading control). Data represent the average ± SD; *p < .05; **p < .01 with respect the control. Figure 3. Open in new tabDownload slide Cycloheximide also induces an apoptotic phenotype and tail-less complex polypeptide-1 eta (TCP1-eta) release, in a concentration-dependent manner. Mouse cortical tubule cells treated with 0 (control), 50, 100, and 200 μM cycloheximide for 18 h. At the end of the experiment, (A) an MTT-based proliferation/viability was performed and (B) the death phenotype was determined by light microscopy, for the presence or absence of cell dismantling into apoptotic bodies (representative images are shown, ×400). The dotted line marks the starting point (time = 0 h). White arrows: apoptotic cells. C, Representative images of Western blot analysis after 18 h of treatment with cycloheximide of active caspase 3 (p19 fragment, n = 2), densitometry quantification, and representative images of Western blot analysis of α-tubulin (as loading control). D, TCP1-eta in the culture medium (n = 3), densitometry quantification, and representative images of Western blot analysis of α-tubulin (as loading control). Data represent the average ± SD; *p < .05; **p < .01 with respect the control. Tail-less Complex Polypeptide-1 eta Is Differentially Elevated in the Urine of Rats With ATN, Correlating With the Degree of Renal Cortical Injury Different rat models of AKI with distinct renal histological damage patterns were used to evaluate whether the secreted TCP1-eta reaches the tubular lumen and appears in the urine as a marker of tubular damage, namely ischemia/reperfusion injury, cisplatin-induced AKI, gentamicin-induced AKI, and prerenal AKI. Plasma creatinine significantly increased in all models (Figure 4A), with increased TCP1-eta (Figure 4B) observed only in the 3 models of ATN (ie, I/R, cisplatin and gentamicin), progressively increasing with increasing damage due to cisplatin (Figs. 4C and 4D). Consequently, no correlation was observed between the urinary excretion of TCP1-eta and the extent of functional damage measured as increments in Crpl (Figure 4E). In contrast, a signature marker of apoptosis (ie, cleaved caspase 3) was not excreted in the urine despite being highly expressed in the kidneys (Figs. 4F and 4G). Figure 4. Open in new tabDownload slide Urinary tail-less complex polypeptide-1 eta (TCP1-eta) is increased in different rat models of intrinsic acute kidney injury (AKI). A, Plasma creatinine (Crpl), (B) representative image of Western blot analysis of urinary TCP1-eta levels, of rats with different types of AKI. Data represent the average ± SEM of n = 4. C, Time course of Crpl and (D) representative image of Western blot analysis of urinary TCP1-eta levels of rats treated with cisplatin. Data represent the average ± SEM of n = 3. E, Correlation analysis of urinary TCP1-eta and Crpl of rats with different types of renal damage. F, Representative image of Western blot analysis of urinary active caspase 3 in rat models of AKI rats (n = 3). G, Representative images (×400) of active caspase 3 immunostaining in the renal cortex of tissue specimens from ischemia/reperfusion (I/R), cisplatin and gentamicin; AU, arbitrary units; B, basal point; D2 and D4, 2 and 4 days, respectively, after cisplatin administration. *p < .05; **p < .01 with respect the control. Abbreviations: PR, prerenal; UNX; uninephrectomy; I/R, ischemia/reperfusion; CISP, cisplatin; GENTAM, gentamicin. Figure 4. Open in new tabDownload slide Urinary tail-less complex polypeptide-1 eta (TCP1-eta) is increased in different rat models of intrinsic acute kidney injury (AKI). A, Plasma creatinine (Crpl), (B) representative image of Western blot analysis of urinary TCP1-eta levels, of rats with different types of AKI. Data represent the average ± SEM of n = 4. C, Time course of Crpl and (D) representative image of Western blot analysis of urinary TCP1-eta levels of rats treated with cisplatin. Data represent the average ± SEM of n = 3. E, Correlation analysis of urinary TCP1-eta and Crpl of rats with different types of renal damage. F, Representative image of Western blot analysis of urinary active caspase 3 in rat models of AKI rats (n = 3). G, Representative images (×400) of active caspase 3 immunostaining in the renal cortex of tissue specimens from ischemia/reperfusion (I/R), cisplatin and gentamicin; AU, arbitrary units; B, basal point; D2 and D4, 2 and 4 days, respectively, after cisplatin administration. *p < .05; **p < .01 with respect the control. Abbreviations: PR, prerenal; UNX; uninephrectomy; I/R, ischemia/reperfusion; CISP, cisplatin; GENTAM, gentamicin. The 3 ATN models demonstrated characteristic patterns of damage, with ischemia affecting mostly the outer medullary strip, cisplatin both the cortex and the outer medulla, and gentamicin mostly the cortex and, to a lesser extent, the medulla. Renal histology and renal damage scores (Figs. 5 and 6) revealed that the level of urinary TCP1-eta tightly correlated with the level of cortical but not of outer medullary damage. Figure 5. Open in new tabDownload slide Urinary tail-less complex polypeptide-1 eta (TCP1-eta) correlates with the level of renal cortical damage. A, Representative images of the cortical area (×400) from kidney specimens stained with hematoxylin and eosin from control, prerenal (PR), ischemia/reperfusion (I/R), cisplatin (CISP), and gentamicin (GENTAM) rats. B, Graphical representation of cortical damage severity score. C, Correlation between urinary TCP1-eta levels and total severity score of cortical damage. Abbreviation: AU, arbitrary units. *p < .05 respect to control; **p < .01 with respect the control group. Figure 5. Open in new tabDownload slide Urinary tail-less complex polypeptide-1 eta (TCP1-eta) correlates with the level of renal cortical damage. A, Representative images of the cortical area (×400) from kidney specimens stained with hematoxylin and eosin from control, prerenal (PR), ischemia/reperfusion (I/R), cisplatin (CISP), and gentamicin (GENTAM) rats. B, Graphical representation of cortical damage severity score. C, Correlation between urinary TCP1-eta levels and total severity score of cortical damage. Abbreviation: AU, arbitrary units. *p < .05 respect to control; **p < .01 with respect the control group. Figure 6. Open in new tabDownload slide Urinary tail-less complex polypeptide-1 eta (TCP1-eta) does not correlate with the level of medullary damage. A, Representative images of the outer medullary area (×400) from kidney specimens stained with hematoxylin and eosin from control, prerenal (PR), ischemia/reperfusion (I/R), cisplatin (CISP), and gentamicin (GENTAM) rats. B, Graphical representation of outer medulla damage severity score. C, Correlation between urinary TCP1-eta levels and total severity score of cortical damage. Abbreviation: AU, arbitrary units. *p < .05 respect to control; **p < .01 with respect the control group. Figure 6. Open in new tabDownload slide Urinary tail-less complex polypeptide-1 eta (TCP1-eta) does not correlate with the level of medullary damage. A, Representative images of the outer medullary area (×400) from kidney specimens stained with hematoxylin and eosin from control, prerenal (PR), ischemia/reperfusion (I/R), cisplatin (CISP), and gentamicin (GENTAM) rats. B, Graphical representation of outer medulla damage severity score. C, Correlation between urinary TCP1-eta levels and total severity score of cortical damage. Abbreviation: AU, arbitrary units. *p < .05 respect to control; **p < .01 with respect the control group. Tail-less Complex Polypeptide-1 eta Is Secreted by Tubule Cells in the Urine and Not Reabsorbed by Damaged Tubules In accordance with in vitro studies, no accumulation of TCP1-eta was detected in kidney cells, by immunohistochemistry (Figure 7A) and Western blot (Figure 7B), in any of the ATN groups. In situ renal perfusion experiments revealed that when the kidneys are perfused with Krebs-dextran solution, a part of the urinary TCP1-eta was eliminated from the urine, but another part remained excreted in the urine (Figs. 7C and 7D). These results indicated that a part of the urinary TCP1-eta appeared from the blood and ended up in the urine because of an altered renal handling (most probably deficient reabsorption); another part was directly shed from renal cells (likely tubule cells) into the urine. Tail-less complex polypeptide-1 eta seemed to be normally filtered and mostly reabsorbed in the proximal tubule, as deduced from 2 facts: (1) under control conditions, no significant, or minimal, TCP1-eta was detected in the urine; and (2) in normal rats treated with sodium maleate, an inhibitor of proximal tubule reabsorption, TCP1-eta was excreted in the urine (Figure 7E). Figure 7. Open in new tabDownload slide Urinary tail-less complex polypeptide-1 eta (TCP1-eta) partly comes from the blood and partly from the renal tissue. A, Representative images (×400) of the cortical area immunostained for TCP1-eta from control, ischemia/reperfusion (I/R), cisplatin and gentamicin. B, Representative image of Western blot analysis of renal tissue TCP1-eta levels and an unspecific band (as loading control) of rats with different types of acute kidney injury (AKI) (n = 3). C, Schematic representation of the experimental set up used for in situ kidney perfusion with Krebs-dextran solution, and urine collection from the bladder. D, Representative image of Western blot analysis of TCP1-eta levels and the corresponding densitometry quantification, in the urine of control and AKI rats during in situ renal perfusion. Data represent the average ± SEM (n = 3). Abbreviations: AU, arbitrary units; B, basal point; **p < .01 versus basal point in its group. E, Time-course of TCP1-eta urinary level in rats treated intravenously with a bolus of sodium maleate (400 mg/kg). Data represent the mean ± SEM. Abbreviation: AU, arbitrary units; **p < .001. Figure 7. Open in new tabDownload slide Urinary tail-less complex polypeptide-1 eta (TCP1-eta) partly comes from the blood and partly from the renal tissue. A, Representative images (×400) of the cortical area immunostained for TCP1-eta from control, ischemia/reperfusion (I/R), cisplatin and gentamicin. B, Representative image of Western blot analysis of renal tissue TCP1-eta levels and an unspecific band (as loading control) of rats with different types of acute kidney injury (AKI) (n = 3). C, Schematic representation of the experimental set up used for in situ kidney perfusion with Krebs-dextran solution, and urine collection from the bladder. D, Representative image of Western blot analysis of TCP1-eta levels and the corresponding densitometry quantification, in the urine of control and AKI rats during in situ renal perfusion. Data represent the average ± SEM (n = 3). Abbreviations: AU, arbitrary units; B, basal point; **p < .01 versus basal point in its group. E, Time-course of TCP1-eta urinary level in rats treated intravenously with a bolus of sodium maleate (400 mg/kg). Data represent the mean ± SEM. Abbreviation: AU, arbitrary units; **p < .001. High Levels of Urinary TCP1-eta Are Associated With Bad Prognosis in AKI Patients Furthermore, urinary TCP1-eta was investigated in patients in the context of AKI. A cohort of etiologically heterogeneous patients, with AKI symptomatology, referred to the nephrology department were included (Figure 8A). They were cataloged as “recovery” or “nonrecovery” depending on whether Crpl returned to values prior to the AKI episode or not, respectively. Healthy controls and disease controls (without AKI) were also evaluated. The urinary level of TCP1-eta was measured at admission. Taken as a whole, the cohort of AKI patients demonstrated higher levels of the urinary marker than healthy and disease controls (Figure 8B). However, there was a wide spectrum of urinary TCP1-eta values within AKI patients ranging from nondetectable to high levels. Controls presented the normal range of variability in the urinary TCP1-eta levels, with a range of nonspecificity (ie, non-AKI-related). Interestingly, urinary TCP1-eta was significantly higher during AKI in patients who did not eventually recover, compared with those who completely recovered. This difference was statistically significant in the whole cohort (Figure 8C), and highly significant among patients at “failure” stage (according to the RIFLE scale; Figure 8D), and among those with intrinsic AKI (according to their Crpl/Cru ratio; Figure 8E). In failure AKI patients and in intrinsic AKI patients, logistic regression analysis demonstrated good correlation between the urinary level of TCP1-eta and AKI evolution (ie, full recovery or nonrecovery) in the whole cohort (Figure 8F). Figure 8. Open in new tabDownload slide Urinary tail-less complex polypeptide-1 eta (TCP1-eta) during acute kidney injury (AKI) predicts patient outcome. A, Study patient characteristics. B, Representative image of Western blot analysis of urinary TCP1-eta levels in each study group, and the corresponding densitometry quantification. Urinary TCP1-eta levels in patients with favorable outcome (ie, total recovery from AKI; Recovery) and with unfavorable outcome (ie, incomplete or no recovery; Non Recovery) in the whole population (C), in patients with failure AKI (according to the RIFLE scale) (D), and in patients with intrinsic AKI (according to their Cru/CrPl ratio) (E). (F) Parameters of the logistic regression analysis for data from C to E. Abbreviation: C/G, Cockcroft-Gault. Figure 8. Open in new tabDownload slide Urinary tail-less complex polypeptide-1 eta (TCP1-eta) during acute kidney injury (AKI) predicts patient outcome. A, Study patient characteristics. B, Representative image of Western blot analysis of urinary TCP1-eta levels in each study group, and the corresponding densitometry quantification. Urinary TCP1-eta levels in patients with favorable outcome (ie, total recovery from AKI; Recovery) and with unfavorable outcome (ie, incomplete or no recovery; Non Recovery) in the whole population (C), in patients with failure AKI (according to the RIFLE scale) (D), and in patients with intrinsic AKI (according to their Cru/CrPl ratio) (E). (F) Parameters of the logistic regression analysis for data from C to E. Abbreviation: C/G, Cockcroft-Gault. DISCUSSION As a reasonable hypothesis, proteins found differentially in the supernatant of tubular cells cultured in vitro with cytotoxic drugs might eventually be found in the urine of treated animals. Based on this approach, ethical concerns on the excessive use of experimental animals might be spared, at least during the first screening stage of biomarker discovery. Our results demonstrated that tubular cells exposed to cisplatin handled TCP1-eta differently, by increasing outflow or decreasing reuptake, which resulted in higher amounts of this protein in the extracellular compartment. The increased outflow may be determined by the increased transcription, as no extra TCP1-eta accumulated inside the cells. The increased extracellular level of TCP1-eta seemed a rather specific event, as the control and cisplatin proteomes in the culture medium are quite similar. This is consistent with apoptotic cells and apoptotic bodies being tight bound and securing the intracellular content. In fact, the absence of a key executor of apoptosis in the culture media, such as the active form of caspase 3, and that of a marker of necrotic rupture of the cell membrane (ie, LDH), indicated that apoptotic mediators are not generally shed in the extracellular space during apoptosis. Accordingly, TCP1-eta has garnered interest and can be further explored as a potential noninvasive marker of cytotoxicity in vitro, which might enable apoptosis monitoring over time by sampling and analyzing culture media. This approach provides specific advantages over end point experiments in which apoptosis is studied in cell extracts for every study time point, with regard to simplicity, resource optimization, and longitudinal consistency. Tail-less complex polypeptide-1 is a chaperonin-containing, hetero-oligomeric complex formed by 8 subunits, including subunit eta. Tail-less complex polypeptide-1 is known to contribute to actin and tubulin folding in the cytosol, and thus to the conformation of the cytoskeleton, cell shape (Doucey et al., 2006; Llorca et al., 2001; Martín-Benito et al., 2002) and cell division (Abe et al., 2009). Tyrosine phosphorylation of TCP1 is also involved in cell detachment during apoptosis (de Graauw et al., 2007). Moreover, as endoplasmic reticulum (ER) protein-folding chaperones, all TCP1 subunits are upregulated upon ER stress (Yokota et al., 2000). Many chemicals and drugs, including cisplatin (Sancho-Martínez et al., 2012), ischemia/reperfusion and other conditions (Taniguchi and Yoshida, 2015) induce ER stress, which in turn activates the unfolded protein response (UPR). Unfolded protein response is a cellular mechanism activated to re-establish ER homeostasis when an excessive level of misfolded proteins accumulate within the ER (Hetz et al., 2015). One of the mechanisms involved in UPR is the overexpression of ER chaperoning proteins involved in correct protein folding. Despite this response, when the level of misfolded proteins and ER stress are not sufficiently reduced, the cell activates the apoptotic program (Hetz et al., 2015). In fact, different renal diseases, including toxic and ischemic AKI are ameliorated when ER stress is prevented (Taniguchi and Yoshida, 2015). In our experiments, TCP1 overexpression may be interpreted as part of ER stress and UPR, which need to be further explored. In particular, the signaling pathways and mechanisms that lead to TCP1 secretion during apoptosis need to be elucidated. Endoplasmic reticulum function and the UPR are tightly connected to the secretory pathways, as a fraction of proteins synthesized in the ER are folded and derived by proteostasis (Plate and Wiseman, 2017). We can only speculate whether TCP1 secretion occurs as a distortion of the secretory endosomal route caused by cisplatin, or as a consequence of apoptosis, or TCP1 is by design secreted as part of the UPR to protect the extracellular environment, as described for other proteins such as ERdj3 (Plate and Wiseman, 2017). Similarly, ERdj3 is an ER HSP40 co-chaperone involved in the UPR, which is secreted in response to ER stress (Genereux et al., 2015) to minimize accumulation of misfolded proteins within the cellular entourage. An implicit hypothesis presented that if tubular cells secrete higher amounts of TCP1-eta into the extracellular space, and if this also happened in vivo, TCP1-eta might reach the ultrafiltrate flowing through the tubular lumen and be found in the urine. In fact, this seems to be the case. Urinary excretion of TCP1-eta was significantly increased in the 3 rat models of intrinsic AKI. Moreover, the level of TCP1-eta urinary excretion correlated with the degree of renal cortical injury rather than with the level of renal dysfunction (measured as Crpl). Interestingly, the in situ kidney perfusion experiments indicated that a part of the TCP1-eta found in the urine in AKI resulted from direct shedding from the kidney (likely tubular) cells, as it remained in the urine during perfusion with Krebs-dextran. However, the other part seemed to be not shed directly by damaged tubule, as it disappeared from the urine during perfusion, to be filtered and undergo altered tubular handling (likely decreased reuptake), as a consequence of tubular damage. Collectively, our results suggested that the damaged cortical tubules both shed TCP1-eta into the urine and were less effective at reabsorbing this protein, as normally performed when unaltered. The mechanisms for bulk protein reuptake is located at the proximal tubule level (Comper et al., 2016), with the renal cortex being enriched in proximal tubules, especially in the convoluted segments. Consequently, the amount of TCP1-eta excreted in the urine depends on the extent of overall damage (a direct source of TCP-1, as shed by damaged tubule cells), and specifically on the extent of cortical damage (responsible for the defective reabsorption of TCP1). In our animal models, as a similar degree of total histological damage was observed (theoretically causing a similar direct shedding of TCP1), it was established that the extent of cortical damage is of value in regards to the total urinary TCP1-eta excretion, as in models with more cortical damage, more TCP1 is expected to escape tubular reabsorption. Importantly, urinary TCP1-eta was clearly associated with AKI in patients, and to worsening prognosis both in the general cohort and AKI population, more robustly when combined with other markers that further substratify the AKI population. Notably, high levels of this marker were associated with insufficient recovery from AKI, considerably within those patients with theoretically worse prognosis, namely patients with “failure” AKI, and those with renal AKI. Based on the knowledge gained in our cell and animal models, high urinary TCP1-eta levels in a heterogeneous population (such as our cohort) may be explained by extensive renal tissue damage, or due to damage more specifically affecting the cortical compartment, or a combination of both, all of which relate to a bad prognosis. In general, biomarkers associated with specific tissue damage patterns or functional alterations will help to further stratify patients into subcategories with objective and quantitative criteria, that may then be more precisely related to diverse clinical outcomes. Accordingly, by incorporating the analysis of TCP1-eta in the urine, AKI monitoring might potentially enhance the gross estimation of worse prognosis anticipated by AKI severity or AKI type (ie, population-based risk analysis), to a finer prognosis based also on TCP1-eta, identifying at the individual level which patients may or may not recover from AKI (ie, truly personalized risk analysis). These results prompt wider clinical investigations, as pre-emptive knowledge may help to improve decision making and patient treatment. In conclusion, this study utilizes a cell-based method for biomarker discovery and identifies TCP1-eta as a urinary marker of renal cortical tubule injury, which appears to be associated with insufficient recovery from AKI in patients. In perspective, this biomarker might potentially contribute to the pathophysiological diagnosis of AKI, allowing a more accurate patient stratification and personalized therapy. DECLARATION OF CONFLICTING INTERESTS The author/authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. FUNDING This study was supported by grants from the Government of Spain (Instituto de Salud Carlos III [PI14/01776, DT15S/00166 and PI15/01055, PI18/00996 and Retic RD016/0009/0025, REDINREN), Ministerio de Economía y Competitividad (IPT-2012-0779-010000]), and FEDER funds. The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration. REFERENCES Abe Y. , Yoon S.-O. , Kubota K. , Mendoza M. C. , Gygi S. P. , Blenis J. ( 2009 ). p90 ribosomal S6 kinase and p70 ribosomal S6 kinase link phosphorylation of the eukaryotic chaperonin containing TCP-1 to growth factor, insulin, and nutrient signaling . J. Biol. Chem. 284 , 14939 – 14948 . Google Scholar Crossref Search ADS PubMed WorldCat Albert C. , Albert A. , Kube J. , Bellomo R. , Wettersten N. , Kuppe H. , Westphal S. , Haase M. , Haase-Fielitz A. ( 2018 ). 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TI - Urinary TCP1-eta: A Cortical Damage Marker for the Pathophysiological Diagnosis and Prognosis of Acute Kidney Injury
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfz242
DA - 2020-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/urinary-tcp1-eta-a-cortical-damage-marker-for-the-pathophysiological-8RJa05jboB
SP - 3
VL - 174
IS - 1
DP - DeepDyve
ER -