Chronic kidney disease (CKD), a serious health problem worldwide arising from multiple causes, is a condition manifested by a progressive decline in renal function associated with irreversible renal pathologies that usually include glomerulosclerosis, vascular alterations, or tubulointerstitial (TI) fibrosis [1]. TI fibrosis, also arising from multiple causes (e.g., acute kidney injury), results from TI accumulation of excess extracellular matrix, primarily composed of collagen, leading to loss of normal renal function. TI fibrosis, also referred to as interstitial fibrosis with tubular atrophy, is an important prognostic factor in several forms of CKD, and is associated with deteriorating renal function [2]. Despite the high prevalence of TI fibrosis in CKD regardless of etiology, there are few biomarkers identified to detect TI fibrosis. Tubular proteinuria, a current method to determine TI injury, does not present at the early phase after tubular injury, and thus its use is not optimal for the early detection of and intervention in TI fibrosis [3].
Identification of alternative biomarkers that would allow early detection of TI fibrosis using a noninvasive approach would offer a significant advantage in its diagnosis. One such approach involves the detection of disease biomarkers that have been released into body fluids as either intact cells or cellular components, a so-called liquid biopsy [4]. Cellular components called exosomes, 30-100 nm membrane vesicles secreted from several cell types, are a rich source of potential biomarkers in urine [5, 6]. Several types of molecules that are easily degraded in the extracellular environment, such as transcription factors and micro-RNAs [6, 7], are enriched and preserved inside exosomes. Therefore, urinary exosomes are a promising source of potential biomarkers of TI fibrosis.
Most current rodent models of CKD target the study of glomerulosclerosis [8]. However, we and others have described studies using a mouse model in which an acute kidney injury (induced by folic acid (FA) injection) results in predominantly TI fibrosis [9, 10] (and references therein). Unfortunately, only small volumes of mouse urine can be obtained, precluding a sufficient yield of urinary exosomes for biomarker discovery experiments. A FA-induced rat model of renal injury is also available and has been extensively characterized with respect to histopathology and renal function [11,12,13,14,15]. Here, we evaluated the FA-induced rat model of renal injury for its suitability in urinary exosome studies to identify biomarkers of TI fibrosis by proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Animal care followed the U.S. National Institutes of Health criteria for the care and use of laboratory animals in research, under protocols approved by the National Institute of Diabetes and Digestive and Kidney Diseases Animal Care and Use Committee (ASP No. K087-KDB-11–14) following guidelines issued by the U.S. Department of Health and Human Services, the U.S. Department of Agriculture, the Association for the Assessment and Accreditation of Laboratory Animal Care, and Office of Laboratory of Animal Welfare (assurance No. A4149-01). Male Sprague Dawley rats (250-300 g) were purchased from Charles River Laboratories (Germantown, MD, USA) and had free access to water and standard laboratory rodent food. Rats were administered FA in 0.3 M NaHCO 3 (FA group, 250 mg/kg body weight) or 0.3 M NaHCO3 alone (vehicle (Veh) group) using a single intraperitoneal injection (0.9 to 1.1 mL) (see Discussion for additional details). While there might be a difference in alkali administration between these groups, (i) the volume of the single injection was small, (ii) any effect on plasma pH would be quite transient, (iii) there was no obvious abnormality in mice after loading of this dose, and (iv) this is the standard protocol for many published studies using the FA-induced model of renal injury. Only FA-injected rats with blood urea nitrogen (BUN) ≥100 mg/dL at day 2 after injection were selected for further study (for animals with BUN <100 mg/dL, the development of TI fibrosis was inconsistent (data not shown).
In standard experiments using this FA-induced model of renal injury, samples of blood (drawn from the tail vein) and urine (24 h, collected using rat metabolic cages; Hatteras Instruments, Cary, NC, USA) were obtained for analysis and body weight was measured, before administration of either FA or Veh (day 0) and at days 2, 7, 14, and 28 after administration. We used 35 rats in the experiments: 20 rats were used to explore the model and 9 were used for the proteomic analysis. The remaining 6 rats failed to produce BUN >100 mg/dL and were excluded from the study.
For quantitative comparisons of putative biomarkers in urine and/or urinary exosomes, both specific protein measurements and normalization based on the total amount of protein in a single urine sample (i.e., a sample collected over a short time) are generally unsatisfactory [16]. First, normal physiological variations in water excretion can lead to relative dilution or concentration of urinary proteins in such samples, thereby affecting the total amount of protein present. Second, total protein excretion in urine can vary broadly among various pathophysiological states. Therefore, for accurate quantitative measurements from urine samples, it is necessary to measure the actual
All urine samples were collected (over 24 h) using metabolic cages, protease inhibitors were added, and samples were centrifuged at 1000 ×
BUN, serum creatinine (SCr), and urine creatinine (UCr) were measured using colorimetric assays (QuantiChrom kits DIUR-500 for urea assay and DICT-500 for creatinine assay; BioAssay Systems, Haywood, CA, USA). Urine protein level (24 h urine albumin) was measured using enzyme-linked immunosorbent assay kits (Nephrat, Exocell, Philadelphia, PA, USA). Because SCr does not reach a steady state during acute kidney injury, we directly calculated creatinine clearance (CCr) from 24 h urine collection of UCr.
The CCr was calculated using the following equation: CCr = (UCr * 24 h urine volume)/SCr. For histology, kidneys were fixed in 10% neutral buffered formalin solution for paraffin embedding and sectioning (4 mm thickness), then stained using a Masson trichrome method. TI fibrosis was evaluated at 200 × magnification by the following semiquantitative criteria to estimate area of damage: 0, <5%; 1, 5%-10%; 2, 11%-25%; 3, 26%-50%; 4, >50%.
For western blot analysis, exosomes from each urine sample were isolated separately; for proteomic analysis, all urine samples from each group were pooled before exosome preparation. Rat urinary exosomes were isolated by differential centrifugation (17,000 ×
After isolation of exosomes from individual rat urine samples, exosomal proteins were separated by one-dimensional SDS-PAGE (12% gel), transferred to nitrocellulose by electroblotting, and probed with the following primary antibodies: mouse monoclonal antibody to ALG-2-interacting protein 1 (ALIX; also known as programmed cell death 6-interacting protein or PDCD6IP) (BD Biosciences, Franklin Lakes, NJ, USA); rabbit polyclonal antibodies to tumor susceptibility gene 101 (TSG101) (Abcam, Cambridge, MA, USA), and clusterin (Sigma Aldrich, St. Louis, MO, USA). Immunoreactivity was visualized with horseradish peroxidase-conjugated secondary antibodies (Sigma Aldrich) and enhanced chemiluminescence substrate (Amersham Biosciences, Little Chalfont, Bucks, UK) using light-sensitive film (Kodak BioMax XAR, Rochester, NY, USA). Band intensities were determined by ImageJ version 1.36b (National Institutes of Health, Bethesda, MD, USA).
Solubilized exosomes from pooled rat urine samples (from 3 rats in each group) were fractionated by SDS-PAGE (12%), digested with trypsin, and analyzed by nanospray LC–MS/MS as described previously [5]. Briefly, each sample lane was cut into eight pieces (from top to bottom), and each gel piece was minced and subjected to in-gel tryptic digestion as follows: washed (25 mM NH4HCO3 in 50% acetonitrile); rehydrated (10 mM dithiothreitol, 25 mM NH4HCO3) at 56°C for 1 h; alkylated (55 mM iodoacetamide in 25 mM NH4HCO3) at room temperature for 1 h; washed twice and rehydrated again at 4°C for 0.5 h; then finally incubated with the sequencing-grade trypsin (product No. V5111; Promega, Fitchburg, WI, USA) at 37°C overnight. Subsequently, peptides were extracted from the gel in 50% acetonitrile containing 5% formic acid, then desalted and concentrated using a ZipTip (Merck Millipore, Darmstadt, Germany), eluted with 0.1% FA and transferred to LC-MS/MS auto-sampler vials. The LC-MS/MS analysis was carried out using a ProteomeX workstation LTQ-MS (Thermo Finnigan, San Jose, CA, USA). The NCBI RefSeq protein database (
All data are expressed as mean ± SEM. Differences between or among groups were analyzed for statistical significance by one-way ANOVA (SPSS for Windows, version 14.0, SPSS Inc., Chicago, IL, USA)
An acute change in renal function, as determined by an increase in BUN concentration and a decrease in CCr, was detected at day 2 after FA injection (
Next, we conducted a preliminary feasibility study to evaluate the use of urinary exosomes to identify novel protein biomarkers of TI fibrosis.
Isolation of exosomes from urine (24 h collection) was confirmed by immunoblot detection of ALIX and TSG101, marker proteins for urinary exosomes [5, 17] (
We performed proteomic analysis of urinary exosomes from rats injected with Veh alone, and at 1 and 2 weeks after FA injection. We identified a combined total of 372 proteins from the exosomes of all three samples; approximately one-third of these proteins (134) were common to all three samples (
Comparison of the most abundant proteins (estimated from normalized spectral counts) identified in urine exosomal samples from vehicle-treated rats and rats 1 and 2 weeks after folic acid (FA) treatment. Membrane protein abundantly/predominantly expressed in the kidney Membrane protein abundantly/predominantly expressed in the kidney Membrane protein abundantly/predominantly expressed in the kidney Membrane protein abundantly/predominantly expressed in the kidney Membrane protein abundantly/predominantly expressed in the kidney Absent in control exosome sample, and present in both 1-week (inflammation) and 2-week (fibrosis) exosome samples from FA-treated rats Absent in control exosome sample, and present in both 1-week (inflammation) and 2-week (fibrosis) exosome samples from FA-treated rats Absent in control exosome sample, and present in both 1-week (inflammation) and 2-week (fibrosis) exosome samples from FA-treated rats Absent in control exosome sample, and present in both 1-week (inflammation) and 2-week (fibrosis) exosome samples from FA-treated rats Absent in control exosome sample, and present in both 1-week (inflammation) and 2-week (fibrosis) exosome samples from FA-treated rats Absent in control exosome sample, and present in both 1-week (inflammation) and 2-week (fibrosis) exosome samples from FA-treated rats Absent in control exosome sample, and present in both 1-week (inflammation) and 2-week (fibrosis) exosome samples from FA-treated rats Absent in control exosome sample, and present in both 1-week (inflammation) and 2-week (fibrosis) exosome samples from FA-treated rats Absent in control exosome sample, and present in both 1-week (inflammation) and 2-week (fibrosis) exosome samples from FA-treated rats Absent in control exosome sample, and present in both 1-week (inflammation) and 2-week (fibrosis) exosome samples from FA-treated ratsRef. Sequence number Protein name Normalized spectral counts Vehicle 1 week post-FA 2 weeks post-FA NP_954887.1 Cystatin-related protein 2 precursor 602 216 403 NP_061998.1 Probasin precursor 373 243 446 NP_446292.1 Gamma-glutamyltransferase 1 282 308 192 NP_598306.1 Common salivary protein 1 195 132 333 NP_037025.1 Actin, gamma-enteric smooth muscle 191 226 314 NP_037347.1 Aflatoxin B1 aldehyde reductase member 3 187 3 24 NP036740.1 Neprilysin 181 101 269 NP_001002807.1 Chloride intracellular channel protein 1 178 62 75 NP_113902.1 Ribonuclease UK114 175 51 66 NP_036767.1 Anionic trypsin-1 precursor 175 362 443 NP_058778.1 Uromodulin precursor 160 70 318 NP_112274.1 Aminopeptidase N precursor 153 109 218 NP_058912.1 Neutral and basic amino acid transport protein rBAT 152 47 132 NP_037315.1 Meprin A subunit beta precursor 115 17 80 NP_997476.1 Prostatic steroid-binding protein C2 112 20 531 NP_036947.1 Glutamate–cysteine ligase catalytic subunit 105 0 17 NP_001005889.2 Radixin 101 39 84 NP_543180.1 ADP-ribosylation factor 3 99 50 39 NP_036850.1 Cystatin-related protein 1 precursor 97 97 608 NP_059001.1 Glutamate–cysteine ligase regulatory subunit 95 7 18 NP_071964.2 Alpha-1-antiproteinase precursor 0 75 178 NP_037264.1 Annexin A5 0 69 47 XP_001055138.1 PREDICTED: similar to putative breast adenocarcinoma marker 0 32 0 NP_444180.2 Clusterin precursor 0 31 22 XP_001055585.1 PREDICTED: similar to grancalcin 0 23 0 NP_01025081.1 Programmed cell death 6 interacting protein (ALIX) 0 21 10 NP_476487.1 Complement component C9 precursor 0 16 0 NP_068611.1 Histone H2A type 4 0 15 8 NP_599153.1 Albumin 0 15 13 NP_620796.1 Galectin-3-binding protein precursor 0 14 7 NP_058821.1 CD63 antigen 0 13 8 NP_059055.2 Ras-related protein Rab-10 0 10 25 NP_001019475.1 Glutathione S-transferase Mu 4 0 9 9 NP_803435.1 Prostatic steroid-binding protein C1 precursor 0 9 18 NP_001004236.1 Tetraspanin-1 0 8 4 NP_071605.2 Complement decay-accelerating factor precursor 0 8 5 NP_073204.1 Transglutaminase 4 (prostate) 0 7 6 NP_446145.1 Cyclin-D-binding Myb-like transcription factor 1 0 7 0 NP_036638.1 Sodium/potassium-transporting ATPase subunit alpha-3 0 7 3 NP_01013087.1 PCTP-like protein 0 7 0 NP_071964.2 Alpha-1-antiproteinase precursor 0 75 178 NP_037264.1 Annexin A5 0 69 47 XP_001070423.1 PREDICTED: hypothetical protein 0 0 45 NP_788265.1 S100 calcium-binding protein, ventral prostate 0 0 33 XP_01055098.1 PREDICTED: similar to carbonyl reductase 3 0 4 29 XP_001068139.1 PREDICTED: hypothetical protein 0 0 27 NP_067599.1 Myoglobin 0 0 26 NP_059055.2 Ras-related protein Rab-10 0 10 25 NP_445937.1 Protein S100-A6 0 0 22 NP_444180.2 Clusterin precursor 0 31 22 NP_037245.1 ATPase, Na+/K+ transporting, beta 1 polypeptide 0 7 20 NP_803435.1 Prostatic steroid-binding protein C1 precursor 0 9 18 NP_001020832.1 Protein crumbs homolog 3 precursor 0 0 18 NP_112313.1 Macrophage migration inhibitory factor 0 0 17 XP_01060853.1 PREDICTED: similar to ATPase, H+ transporting, lysosomal Accessory protein 2 0 0 16 NP_036792.2 Elongation factor 1-alpha 2 0 2 15 NP_001013169.1 Guanine nucleotide-binding protein subunit alpha-14 0 0 14 NP_446316.1 Transitional endoplasmic reticulum ATPase 0 2 14 NP_071636.1 Brain acid soluble protein 1 0 0 14 NP_068512.1 Monocyte differentiation antigen CD14 precursor 0 0 13
Among the most abundant proteins identified uniquely in urinary exosomes from FA-treated rats (i.e., not present in control exosomes), the top 2 most abundant proteins (-1-antiproteinase and annexin A5) were common to both 1- and 2-week FA samples (Table 1). In addition, increased abundance in urine following acute kidney injury has been demonstrated previously for both of these proteins [23, 24]. With respect to potential use of urinary exosomes for biomarker discovery, the results for one of these proteins, annexin A5, were particularly interesting. In all, proteomic analysis identified 8 different annexin proteins, which could be grouped into 3 categories: (i) those found in relatively moderate abundance in all 3 samples; (ii) those found in low abundance and only in some samples; and (iii) annexin A5, which was absent in control exosomes, but was found in moderate abundance in both exosome samples from FA-treated rats (Table 2). This result is consistent with previous identification of annexin A5 as a potential urine biomarker for diseases of the kidney [24].
Annexin family proteins identified in rat urine exosomesRefSeq number Protein name Normalized spectral counts Vehicle 1 week post-FA 2 weeks post-FA Group 1: found in relatively moderate abundance in all 3 samples NP_001011918.1 Annexin A11 56 91 111 NP_077069.3 Annexin A4 50 41 9 NP_063970.1 Annexin A2 18 74 29 Group 2: found in low abundance and only in some samples XP_001071546.1 PREDICTED: similar to Annexin A10 9 6 0 NP_037036.1 Annexin A1 6 6 0 XP_001068572.1 PREDICTED: similar to annexin A13 6 0 3 NP_569100.1 Annexin A7 2 13 2 Group 3: found in moderate abundance only in exosome samples from FA-treated rats NP_037264.1 Annexin A5 0 69 47
Another interesting protein identified uniquely in urinary exosomes from FA-treated rats was clusterin (Table 1 and Supplemental Data, which has been previously reported as a biomarker for acute kidney injury [25] and renal fibrosis [26]. In the present study, we confirmed the proteomic result of significantly increased clusterin after FA administration by immunoblotting (
Acute tubulointerstitial (TI) injury, leading to subsequent TI fibrosis, is responsible for a significant number of cases of acute and chronic kidney diseases encountered in clinical practice [27, 28]. At present, reliable early diagnosis of TI fibrosis by current clinical and laboratory techniques is not possible [29]. Urinary biomarkers that would allow early detection of TI fibrosis using a noninvasive approach would be a significant advance in clinical diagnosis and monitoring of CKD. While analysis of urinary exosomes using large-scale proteomics seemed to be an ideal strategy for discovery of the requisite biomarkers, this approach requires identifying a suitable animal model of TI fibrosis.
Rodent models of acute kidney injury and TI fibrosis, involving a single injection of a large dose of FA, were first described almost 50 years ago [11, 30] and then extensively characterized over the following 2 decades [12,13,14,15]. Major findings that have been reported after numerous studies are that the acute kidney injury after FA injection is (i) dose dependent, (ii) almost exclusively limited to renal collecting ducts, and (iii) no other tissues appear to be affected [9, 11, 14, 30].
Following the protocol described in the original report [11], virtually all of the subsequent studies employing the rat model have used administration of FA by intravenous injection. This often leads to accumulation of sharp, needle-like FA crystals in renal tubules, resulting in physical/obstructive damage to the tissue, which was originally thought to be the mechanism responsible for renal toxicity. However, Fink et al. [15] showed that the crystal formation could be markedly reduced/eliminated by intravenous injection of FA in rats pretreated with NaHCO3 to induce alkalosis (increasing the solubility of FA), but the injury to the renal collecting ducts persisted, thereby demonstrating a direct nephrotoxic effect of FA on these cells. Furthermore, by contrast with studies using the rat model, intraperitoneal injection of FA is employed in studies using mice [9, 30] and FA crystal formation in renal tubules has never been reported, yet acute kidney injury and fibrosis still occurred exclusively in the collecting ducts. After nearly 30 years, the mechanism of this toxicity remains unknown, but is still under active investigation.
The majority of investigations using this rat model of renal injury have been short-term (2-4 days) focused on mechanisms of cell proliferation, cell death, tissue regeneration, or hypertrophy. In contrast, for the present study we used this model to focus specifically on TI injury and fibrosis over a much longer time period (up to 4 weeks). Another novel aspect of the present study using the FA-injected rat model is our adoption of the intraperitoneal route for injection of FA, to avoid crystal formation in the renal tubules.
We injected FA intraperitoneally in rats and characterized the model, identifying an early TI inflammatory phase (apparently maximal after 1 week) accompanied by the slightly delayed development of a fibrotic phase (reaching a maximum between 1-2 weeks). Most indicators of renal function returned to normal within 1-2 weeks. Next, as a proof of concept for biomarker discovery, we determined that a 24 h urine collection using this FA-induced rat model of TI fibrosis was adequate for the urine exosome proteomic analysis. Using proteomic LC-MS/MS analysis in this preliminary evaluation study, we identified numerous exosome-associated and kidney-expressed proteins, confirming both the nature and origin of the proteins we had isolated from urine. As further confirmation, proteomic analysis of samples isolated from rats after renal injury and development of TI fibrosis revealed the presence of elevated levels of known exosome-associated proteins, and 132 other proteins that were not detected in the Veh-treated control rats (from a combined total of 372 proteins identified in all samples). Among this latter group of 132 proteins were at least two proteins that have been suggested previously as biomarkers of renal injury, including clusterin [25] and annexin A5 [24, 31]. Urinary exosomes could be secreted from all cell types lining the urinary tract system (e.g., podocytes, tubular cells, and ureter, and bladder epithelial cells). Based on cross-referencing with the rat transcriptomic databases from microdissected rat glomeruli and tubule segments [32], we found that most of the exosomal proteins found from urine of FA-treated rats at 1 week or 2 weeks cannot be confidently assigned to a particular cellular origin; however, Alas2, Kcnmb2, and Lrg1 were uniquely mapped to glomerular origin, and Serpina3n and Cubn were uniquely mapped to tubular origin.
Further studies are needed to confirm and explore these initial findings, and to identify a collection of biomarkers that exhibit variation in presence or abundance in urine exosomes at various times after renal injury and development of TI fibrosis. Dynamic profiling of such biomarkers may provide clinicians with useful tools with which to diagnose, monitor, and predict prognosis of TI fibrosis at different stages of kidney diseases. This rat model that produces such rapid TI fibrosis, occurring within 2 weeks after FA injection, should also be quite useful for future research in the field of renal injury and CKD.