The kidney’s proximal tubules (PTs), when healthy, reabsorb the majority of total glomerular filtrate, including large quantities of water, electrolytes, and nearly 100% of filtrate protein. Bulk reabsorption is enabled by the extensive surface area of apical microvilli, the so-called brush border, and their extensive reabsorption apparatus. Loss of the PT brush border has been reported in both acute kidney disease and CKD (1,2), and the pathologic effects of progressive brush border loss on renal function has been established in a mouse model of acute-to-CKD transition (3). The rat 5/6 nephrectomy (5/6Nx) remnant kidney model of CKD recapitulates many elements of human CKD, including brush border loss, proteinuria, albuminuria, polyuria, and more (1,3,4). We have observed the timing of brush border loss in these rats to coincide with renal functional decline, and with the presence of large extracellular vesicles originating from PTs (1). This process is distinct from AKI-associated brush border necrosis and sloughing. We term these vesicles large renal tubular extracellular vesicles (LRT-EVs). Despite our identification of what could be LRT-EVs published as histologic representations by other groups (with the earliest found dated 1914), we were unable to find explicit, in-text, literature reference to them, rendering their identification and functional consequence uncertain. LRT-EVs are too large to be exosomes (40–100 nm) or microvesicles (100–1000 nm) (5). Although the LRT-EV size range is compatible with apoptotic bodies (800–5000 nm) (5), there is no evidence of PT epithelial apoptosis via terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL) staining, and other methods, in our 5/6Nx model during the period of LRT-EV production.
CKD now afflicts >15% of the US adult population (6). New diagnostic and treatment strategies for CKD are urgently needed. Assessment of urine-excreted biomarkers has long been a diagnostic and prognostic cornerstone of kidney disease. Rapid technologic advances are changing this field. Old standbys such as urine albumin are now being supplemented by new-age, “omics-based” (e.g., transcriptomics), urine-excreted biomarker analysis. Many such studies focused on extracellular vesicles, especially exosomes, and microvesicles, and provided novel insight into renal pathophysiology. We know the presence of our CKD rat LRT-EVs to temporally coincide with marked exacerbation of renal pathology (1). On the basis of these observations, we hypothesized that LRT-EVs are excreted in the urine and contain proteins essential to healthy renal function. Toward this, and while mindful of insightful omics-based extracellular vesicle analysis from others, we performed a tandem mass spectrometry (MS/MS) proteomic analysis of LRT-EVs we isolated from CKD rat urine. We found LRT-EVs to contain a wide array of tubule proteins. Many of these were PT epithelial proteins that, upon disruption, are known to contribute to hallmarks of renal disease. This study provides needed information upon hitherto poorly characterized, renal pathology–associated LRT-EVs. Because LRT-EVs contain proteins essential to healthy renal function, their formation and excretion represents a potential, newly described mechanism of renal pathology.
Materials and Methods
All animal protocols were approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee and performed in compliance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (Envigo, Madison, WI) were maintained on 0.4% sodium chloride chow (AIN-76A Purified Rodent Diet; Dyets Inc., Bethlehem, PA) and water ad libitum, under a 12-hour light cycle. At 10 weeks of age, animals underwent a sham or 5/6Nx operation, as described previously (1,4). In brief, rats were anesthetized (50 mg/kg ketamine, 8 mg/kg xylazine, and 5 mg/kg acepromazine), and the entire right kidney and left kidney poles were removed by surgical excision. Gelfoam coagulant was applied to resected surfaces. All subsequent rat data collection was performed 10 weeks after the 5/6Nx or sham surgery.
Tissue Collection and Histology
Tissues were excised from anesthetized animals and immediately immersion fixed in 10% formalin. Kidneys were processed, paraffin-embedded, sectioned (4 μm), and stained with Masson trichrome by the Children’s Research Institute Histology Core at the Medical College of Wisconsin. Light microscopy was performed using an Eclipse E-400 microscope (Nikon). TUNEL staining was performed following the manufacturer’s instructions (Click-iT Plus TUNEL Assay), with DNAase I pretreatment as a positive control (Thermo Fisher). Mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories) was used to label nuclei. Imaging was performed on a Nikon A1-R laser scanning confocal microscope. Immunohistochemistry for megalin was performed as previously described (7), with 1:50 megalin antibody (AB184676; Abcam) and 1:100 Texas Red–labeled anti-mouse antibody (T-862, 1:100; Invitrogen). Immunohistochemistry for sodium-hydrogen antiporter 3 (NHE3; also known as SLC9A3) was performed in the same manner, except with 1:50 NHE3 antibody (MABN1813; Millipore Sigma). Imaging was performed with an Eclipse 80i microscope (Nikon) or Nikon A1-R confocal microscope. Histologic sections of PTs, stained with Masson trichrome, from sham and 5/6Nx rats were assessed for the presence of LRT-EVs. This analysis was performed on histologic sections acquired from separate cohorts of sham and 5/6Nx rats at 2, 4, 5, 7, and 10 weeks postsurgery.
LRT-EV Isolation and Diameter Measurement
Sham-operated and 5/6Nx rats (n=3 per group) were housed in wire-bottom metabolic cages, and urine was collected into ice-immersed conical tubes for 24 hours. All subsequent steps were performed at room temperature. Urine was filtered (70-µm mesh), and then centrifuged at 300 × g for 15 minutes (spin A). The resulting pellet was resuspended in 1 ml PBS and centrifuged again (spin B). The supernatant from both spins (A and B) was combined and centrifuged at 650 × g for 10 minutes (spin C), to remove any larger material or shed cells. The supernatant from spin C was centrifuged at 2000 × g for 10 minutes (spin D), to pellet the LRT-EV’s. The pellet was then washed in 1 ml of PBS and centrifuged at 580 × g for 10 minutes (Spin E). An aliquot of this final isolate was inspected and imaged under light microscopy for the presence of LRT-EVs. No LRT-EVs were observed in samples fractionated from sham-operated rat urine. The LRT-EV isolation methodology is summarized in Supplemental Figure 1. The size of LRT-EVs from 5/6Nx rats was measured from the light-microscopy images using the Analyze Particles feature of FIJI software (free download: https://imagej.net/Fiji/Downloads).
Proteomic Sample Preparation and Analysis
The remaining LRT-EVs were transferred to a Dounce homogenizer and mechanically lysed on ice, followed by 30 minutes of water-bath sonication at room temperature, as previously described (8). Homogenized LRT-EVs were then buffer swapped (5×) and concentrated into 250 μl of 25 mM ammonium bicarbonate, pH 8.0, using 3K MWCO Amicon Ultrafiltration Units (Millipore), as described by the manufacturer’s protocol. The concentrated LRT-EV protein fractions then had 0.1% of the MS-compatible RapiGest surfactant (Waters) added, followed by incubation on ice for 10 minutes. Samples were then reduced (10 mM dithiothreitol for 30 minutes at 37°C), alkylated (20 mM iodoacetamide for 30 minutes in the dark at room temperature), and digested with Trypsin Gold (Promega), as previously described (89–10). After protein digestion, all samples were desalted/concentrated using OMIX C18 zip-tips (Varian), according to the manufacturer’s protocol, and prepared for liquid chromatography–MS/MS analysis as previously described (89–10). Peptides were separated on a NanoAcquity UPLC system (Waters) with a C18 (Phenomenex) column with a 240-minute gradient (2%–98% acetonitrile) and analyzed with a LTQ-Orbitrap Velos MS (Thermo Fisher), as previously described (8,10,11).
The MS/MS spectral data were searched against the rodent UniProtKB protein databases in Mascot and SEQUEST algorithms, followed by comparative analysis using the Visualize proteomic software (8,12). Within the software, preference was given to rat, and the Mascot/SEQUEST matches were combined for each run using the combine search function, which matches each scan to the best spectral match from either algorithm to ensure no redundancy. Filters were applied for each search, including a Visualize P>0.85 (false discovery rate<5%). All biologic replicates (N=3 with two technical replicates of each; six total runs) were then combined in Visualize where a peptide filter of two or more peptides and a scan count filter of six was applied. The unique proteins detected in the experiments were uploaded to Ingenuity Pathway Analysis, using the UniProt accession number for subsequent categorization of proteins by known cellular location and function/type (7). The MS proteomics data have been deposited to the ProteomeXchange Consortium, via the PRIDE partner repository, with the dataset identifiers PXD019207 and 10.6019/PXD019207 (13).
Histologic assessment for the presence of LRT-EVs in PTs (Figure 1D) are represented as average±SEM. These data were not normally distributed, as assessed by the Shapiro–Wilk test. Therefore, the nonparametric Mann–Whitney test was used to determine the presence of significant differences (P<0.05) between groups.
We observed vesicles budding from PTs 10 weeks after 5/6Nx surgery (Figure 1A), a time when these rats exhibit substantial pathology, including tubular damage, proteinuria, and cardiovascular dysfunction (1). Sham-operated rats had intact brush borders and lacked lumenal vesicles. In 5/6Nx rats, the PT epithelial cells in regions of large vesicle production had intact nuclei upon visual inspection, were not TUNEL positive, and no TUNEL or DAPI signal was detected within the vesicles (Figure 1B), suggesting these vesicles are not apoptotic bodies. We isolated LRT-EVs (Figure 1C) from 5/6Nx rat urine using a purpose-developed, filtration and differential centrifugation methodology (Supplemental Figure 1). We histologically determined the presence of LRT-EVs in PTs in sham and 5/6Nx rats at 2, 4, 5, 7, and 10 weeks postsurgery (Figure 1D). The presence of LRT-EVs in PTs was time dependent. At 2 weeks postsurgery, 2%±1% (average±SEM) of 5/6Nx PTs had LRT-EVs; at 4 weeks, 8%±3%; at 5 weeks, 7%±3%; at 7 weeks, 51%±6%; and at 10 weeks, 59%±5%. The PTs of sham-operated rats had virtually no LRT-EVs at any of those time points. Microscopic image analysis of LRT-EVs isolated from urine at 10 weeks postsurgery revealed them to be circular in shape (in the two-dimensional images), with an average diameter ± SD of 2.8 ± 1.5 µm, and median diameter of 2.5 µm. It is possible that LRT-EVs of a larger diameter were eliminated as part of the isolation methodology. LRT-EV size and protein markers render them distinct from exosomes, microvesicles, and apoptotic bodies (Figure 1E).
Table 1. -
Summary of protein diversity in large renal tubular extracellular vesicles
||Entrez Gene Name
||Glutamate-cysteine ligase catalytic subunit
||Voltage dependent anion channel 2
||Voltage dependent anion channel 1
||Triokinase and FMN cyclase
||Phosphoglycerate kinase 1
||Phosphoenolpyruvate carboxykinase 1
||Phosphoglycerate mutase 1
||Acid phosphatase 2, lysosomal
||5′-Nucleotidase, cytosolic 1B
||Fatty acid binding protein 9
||ATP synthase peripheral stalk-membrane subunit b
||ATP synthase F1 subunit β
||Secreted phosphoprotein 1
||Myeloid-derived growth factor
||Serpin family A member 1
||Serine protease inhibitor A3K precursor
||Ectonucleotide pyrophosphatase/phosphodiesterase 3
||Cell division cycle 42
|G protein–coupled receptors
||G protein–coupled receptor class C group 5 member C
||Chloride intracellular channel 4
||Calcium voltage-gated channel subunit α1 I
||Transient receptor potential cation channel subfamily M member 2
||SLC9A3 regulator 1
||Alanyl aminopeptidase, membrane
||Dipeptidyl peptidase 4
||LDL receptor-related protein 2/megalin
||Solute carrier family 3 member 1
||PDZ domain-containing 1
||Polymeric Ig receptor
||Solute carrier family 6 member 18
||Solute carrier family 15 member 2
||Solute carrier family 23 member 1
||ATP binding cassette subfamily G member 2
||ATPase H+ transporting V1 subunit A
This table summarizes the three most abundant proteins (by scan count and if present) within each functional category (type) of select cellular locations identified by Ingenuity Pathway Analysis. The ten most abundant transport proteins in the plasma membrane are indicated. UniProt accession numbers, protein symbols, and Entrez gene name are also provided. See Supplemental Table 1
for a complete list of Ingenuity Pathway Analysis–identified protein location/types. SLC9A3, sodium-hydrogen antiporter 3; FMN, flavin mononucleotide; H+
, hydrogen ion.
Proteomic analysis of isolated LRT-EVs detected 447 proteins (Supplemental Table 1). These proteins were of diverse functionality and derive from numerous cellular locations (Figure 2A). Of the total proteins, 46% were cytoplasmic proteins, 24% were extracellular space proteins, 7% were nuclear proteins, and 20% were plasma membrane proteins. Proteins within these respective cellular domains were subdivided by functionality. Subgroups included the following: transporters, enzymes, ion channels, kinases, peptidases, transcriptional regulators, phosphatases, G protein–coupled receptors, cytokines, and transmembrane receptors (Table 1). The LRT-EVs contained megalin (LDL-related protein 2; LRP2) in relatively high quantities. Megalin is a PT-microvilli reabsorption mediator of numerous ligands, including vitamin carrier proteins, lipoproteins, hormones, enzymes, immune-related proteins, and select drugs and toxins (16). Consistent with the proteomics, immunohistochemical analysis indicated the presence of megalin within LRT-EVs (Figure 2B). We also observed that megalin was abundant throughout the PT in 5/6Nx rats, but its intracellular localization was abnormally distributed. NHE3 (SL9A3) is a PT protein that was not identified in LRT-EVs. Consistent with these data, immunohistochemical analysis indicated robust NHE3 expression in sham-operated rat PT epithelial cells, reduced expression in PT epithelial cells of 5/6Nx rats, and no expression in LRT-EVs (Figure 2C).
We demonstrate there to be innumerable, protein-laden, LRT-EVs in the PTs and urine of 5/6Nx rats 10 weeks postsurgery. LRT-EVs house proteins essential to PT reabsorption. The importance of PT brush-border reabsorption is made clinically evident by the severity of the genetic diseases in which it is comprised (e.g., Dent disease, Fanconi syndrome). The loss of numerous functionally important proteins through LRT-EV formation in a CKD-like model of chronic renal stress/insufficiency has not been characterized. These shed proteins are varied in function, and, therefore, their loss likely contributes to a spectrum of CKD-related phenotypes. The effect size of these lost proteins on cellular function is likely dependent on a complex relationship between the magnitude of protein loss, cellular demand for that protein’s function, and the cell’s ability to compensate through translational replacement and/or compensatory mechanisms. Loss of PT proteins has been described in other models of renal injury (3,17). It is possible this protein loss is caused, in part, by LRT-EV formation and excretion, although additional studies would be required to confirm this.
LRT-EV size, lack of protein markers specific to other extracellular vesicles, and presence only in specific pathologic settings, render them distinct from relatively well-characterized urine extracellular vesicles such as exosomes, microvesicles, and apoptotic bodies. To the best of our knowledge, this is the first, explicit description of LRT-EVs. Although much work remains to more fully characterize LRT-EVs, this study enables us to draw several inferences about them. First, LRT-EVs are likely derived, at least in part, from the PT epithelium. This is suggested by their absence in the Bowman’s capsule; their presence in the PT; their temporal correspondence to PT brush-border loss; and their containment of many PT-specific proteins, including aquaporin 1 (AQP1), cubilin, and megalin. Second, LRT-EV presence in CKD 5/6Nx rats, but not in sham-operated rats, may suggest LRT-EVs lack membership within the “healthy” tubule extracellular vesicle milieu, but rather exist consequent to pathology. In this respect, LRT-EVs bear resemblance to oncosomes and exophers. Oncosomes are very large extracellular vesicles (1–10 µm), carry oncogenic proteins, and have only been observed in association with cancer (18,19). Exophers, which are also large (4 µm), are extracellular vesicles produced by Caenorhabditis elegans neurons only under specific conditions of neuronal stress. Exophers appear to be a means by which these neurons package and jettison neurotoxic components (20). Not only are LRT-EVs similar to oncosomes and exophers in size, but also in their derivation from a particular tissue type only under specific pathologic conditions. In contrast, exosomes and microvesicles are found in urine from healthy individuals. Third, urine exosomes, microvesicles, and apoptotic bodies are each associated with specific protein markers. These markers often reflect the mechanism of their respective vesicle’s formation. For example, exosomes are endosomal in origin and contain protein markers (e.g., TSG101, ALIX) associated with the endosomal system (5,14,15). Similarly, microvesicles form through outward budding of the plasma membrane; some proteins involved in this process, such as ARF6, are microvesicle protein markers (14). Apoptotic bodies are associated with markers of apoptosis, such as caspase 3 (14,15). The protein markers of exosomes, microvesicles, and apoptotic bodies were largely absent from LRT-EVs, perhaps suggesting LRT-EVs have a formation mechanism distinct from those extracellular vesicles.
Many LRT-EV proteins are important for maintenance of cellular homeostasis (Figure 3A). For example, regucalcin is an important nuclear and cytoplasmic calcium ion regulator. Superoxide dismutase family members SOD1 and SOD3 are critical to the cellular response to reactive oxygen species. Voltage-dependent anion-selective channels VDAC1 and VDAC2, and ATP synthase subunits (ATP5F1A, ATP5F1B, ATP5PB) play key roles in mitochondrial energetics. Loss of such proteins may reduce the PT’s capacity for reabsorption, albeit indirectly. LRT-EVs also contained many proteins directly involved in reabsorption (Figure 3B). Their collective loss likely contributes to a spectrum of CKD-related phenotypes. Our proteomic analysis identified 16 proteins of the solute carrier family—plasma membrane transporters that facilitate reabsorption of many solutes. Among them were SLC3A1 and SLC34A1, functional loss of which is associated with cystinuria and phosphaturia, respectively (21,22). Proteomics identified sodium-glucose cotransporters (SLC5A1 and SLC5A2) in LRT-EVs. Their functional inhibition is associated with glucosuria, which is reported to occur with progressive brush-border loss (3). Megalin and cubilin, both found within LRT-EVs, are receptors that facilitate reabsorption of a wide variety of ligands, including proteins. LRT-EVs contained five subunits of vacuolar ATPase (ATP6V1A, ATP6V1B2, ATP6V0D1, ATP6V0C, and ATP6V1E1), a proton pump that is in PT epithelia and in distal tubule intercalated cells (23). Vacuolar ATPase regulates organelle pH and endocytic reabsorption of filtered proteins, among other functions (24,25). Also lost in urine-excreted LRT-EVs was G protein–coupled receptor family C group 5 member C (GPRC5C). Gprc5c-knockout mice have reduced blood pH and elevated urine pH (26). LRT-EVs contained AQP1, an important mediator of water balance. AQP1 loss impairs the kidney’s ability to concentrate urine (27). LRT-EVs contained sodium-potassium ATPase (Na+/K+-ATPase) subunit α 1, a primary component of the basolateral Na+/K+-ATPase. The transmembrane Na+/K+-ATPase is responsible for generating the electrochemical gradient that facilitates virtually all energy-dependent tubular reabsorption in the PT.
Some PT proteins abundantly expressed in health were identified in LRT-EVs (e.g., SLC2A1 and SLC34A1), whereas others were not, such as SLC9A3 (NHE3). This could indicate that inclusion of specific proteins into LRT-EVs is not strictly on the basis of abundance. It may also reflect intrinsic protein-expression changes consequent of pathology. For example, NHE3 expression, highly expressed in health, is significantly reduced only 2 weeks after 5/6Nx in rats (28,29). Our immunohistochemistry analysis of NHE3 expression (Figure 2C) is consistent with these previous studies. Thus, LRT-EVs may lack NHE3, and other PT proteins abundantly expressed in health, due to a pathology-related reduction in expression. Interestingly, NHE3-regulator 1 (NHE3R1) was identified in LRT-EVs. NHE3R1 is involved in NHE3 intracellular trafficking (30). Thus, loss of NHE3R1 to LRT-EVs may have contributed to the differences in NHE3 expression between sham and 5/6Nx rat PT epithelia. From this study, we are unable to determine how and why specific proteins were included in LRT-EVs while others were not. Inclusion likely depends upon a complex amalgam of regulated and stochastic factors.
Several studies have made important contributions to our knowledge of the PT and urinary proteome (3132–33). The “shotgun” proteomics-based analysis of 5/6Nx LRT-EVs provided here adds to our understanding of renal tubular pathophysiology. Hypothesis-driven investigations of LRT-EV production and characteristics in different disease states and model systems may provide deeper understanding of changes that occur during CKD progression. This study had a number of limitations. First, the marked increase in LRT-EV formation and excretion that occurred between week 5 and 7 postsurgery coincided with the timing of a sharp decline in renal function in this model, including elevations in proteinuria, and an abrupt increase in histologic evidence of proximal tubule pathology, including hypertrophy and dilation (1). Despite this, we are unable, from this study, to determine conclusively that LRT-EVs are a pathogenic mechanism of CKD. However, the fact that they house functionally important tubule proteins suggests they are, at a minimum, formed during a period of rapid worsening of renal function in the 5/6Nx model of CKD. Second, many LRT-EV proteins were specific to the PTs, but others were not. For example, LRT-EVs contained FABP3, which, in health, is expressed in greater abundance in distal tubules than PTs (34), and is considered a urinary biomarker of distal tubule damage (35). Although PTs appear to be the predominate site of LRT-EV formation, LRT-EVs may be generated at additional sites. Third, we performed our proteomic analysis on LRT-EVs collected from 5/6Nx rats 10 weeks postsurgery. The LRT-EV proteome likely varies with renal injury type and with advances (or retreats) in disease progression.
In conclusion, the 5/6Nx rat model of CKD spontaneously produces LRT-EVs that are excreted in the urine. Our proteomic analysis indicates LRT-EVs contain a wide assortment of proteins, including those specific to the PT epithelium such as transporters, enzymes, transmembrane receptors, and endocytic receptors (Figure 4). This study adds to the rapidly growing field of omics-based, renal disease, extracellular-vesicle analysis. Therapies designed to limit LRT-EV formation may improve renal function. Loss of important tubule proteins via excretion of LRT-EVs may represent an underappreciated pathogenic mechanism of renal disease.
All authors have nothing to disclose.
This research was supported by American Heart Association grant 13SDG17100095, NIH National Center for Advancing Translational Sciences grant 8UL1TR000055, and NIH National Heart, Lung, and Blood Institute grant R01HL128332 (to A. Kriegel).
This article contains the following supplemental material online at http://kidney360.asnjournals.org/lookup/suppl/doi:10.34067/KID.0001212020/-/DCSupplemental.
Supplemental Table 1. List of LRT-EV proteins detected by our proteomic analysis and their IPA-identified cellular location and function.
Supplemental Figure 1. LRT-EV isolation methodology.
R. Adam, B. Hoffmann, and A. Kriegel wrote the original draft and were responsible for formal analysis and validation; R. Adam and A. Kriegel were responsible for data curation; R. Adam, A. Kriegel, M. Paterson, and L. Wardecke were responsible for methodology; R. Adam and L. Wardecke were responsible for investigation; A. Kriegel conceptualized the study and was responsible for funding acquisition, project administration, supervision, and visualization; and all authors reviewed and edited the manuscript.
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