Tissue injury can result from various stimuli, including infections, autoimmune reactions, toxins, radiation, and trauma. The repair process after parenchymal injury typically involves a regenerative phase, in which injured cells are repaired or replaced, without leaving a lasting evidence of damage.1 2 3 , 4
Kidney fibrosis is responsible for most types of chronic progressive kidney failure, affecting approximately 8%–10% of individuals in the developed world.5 , 6 7 , 8 9 – 11 12
Exosomes are small extracellular membrane vesicles secreted by various cell types, including platelets,13 14 15 16 17 18
Prominent kidney fibrosis induced due to unilateral ureteral obstruction (UUO) is observed by day 10 after surgery, with accumulation of myofibroblasts seen as early as day 2 after UUO. End stage fibrosis is mainly associated with fibroblast activation and collagen I deposition.4 , 19 19 , 20 α -smooth muscle actin (α -SMA) (day 2 of UUO) (Figure 1A ). As early as day 2, post-UUO, wrinkling and rupture of the tubular basement membrane is observed, as demonstrated by discontinuous laminin staining (Figure 1B ). Such basement membrane disruption may provide easy transit route for exosomes from injured TECs to the interstitial space. We isolated, characterized, and quantified exosomes from contralateral control kidneys and fibrotic UUO kidneys using transmission electron microscopy (TEM), Western blotting (using CD63 as an exosome marker), and total protein quantification (Figure 1, C–E ). On day 2 after UUO, these kidneys exhibited increased exosome production compared with control kidneys, as measured by CD63 expression and protein quantification (Figure 1, D and E ). In addition, we demonstrate that fibrotic kidneys secrete exosomes highly enriched in TGF-β 1 mRNA compared with contralateral control kidneys (Figure 1F ).
Figure 1:
An increased number of exosomes are released by fibrotic kidneys that contain TGF-β 1 mRNA. (A) Hypoxyprobe, bromodeoxyuridine, and α -SMA staining in kidney cortex after 2 days of UUO surgery using the contralateral kidney as control. Graphs on the right represent the respective quantifications using the number of positive intersections per field or the number of positive nuclei. (B) Immunofluorescence staining with laminin antibody at UUO day 2. Arrows point to the site of tubular basement membrane disruption. (C) Morphologic characterization using electronic microscopy of exosomes extracted from whole contralateral or UUO kidneys of operated mice. The size of exosomes is quantified and expressed in nanometers. (D) Protein blot of exosomes extracted from contralateral and UUO kidneys of operated mice using CD63 antibody and β -actin as loading control with densitometry quantification. Protein extracts prepared from exosomes are normalized by protein extract volume/tissue weight. (E) Protein extracts prepared from exosomes are normalized by protein extract volume/tissue weight and expressed in micrograms per milligram of weight. (F) TGF-β 1 mRNA content in exosomes extracted from mouse kidneys after 2 days of UUO. Results are depicted in percentages of TGF-β 1 mRNA expression in UUO compared with CL kidney. CL, kidney from contralateral side. UUO, kidney after UUO surgery. Original magnification, ×200 in A. *P <0.05.
Mouse and human TEC lines, MCT and HK2, were cultured under normoxic and hypoxic conditions for 24 and 48 hours. A significant increase in hypoxia inducible factor 1α (HIF-1α ) protein was observed after 24 and 48 hours of hypoxia (Figure 2A ). Lactate production was also significantly increased in TECs after 48 hours of culture in hypoxic conditions (Figure 2B ). Exosomes were extracted from TECs and evaluated by TEM. Nanovesicles with a size of ≤100 nm were identified (Figure 2C ). To evaluate the number of exosomes released by TECs in hypoxic and normoxic conditions, relative total protein content was measured and expression of membrane-associated CD63 protein was assessed17 Figure 2D and Supplemental Figure 1, A and B). Expression of CD63 was higher in exosome preparations from hypoxic TECs at 24 and 48 hours compared with normoxic TECs (Figure 2D blot and Supplemental Figure 1A). Total exosome protein normalized by cell number was also significantly higher when isolated from hypoxic TECs (Figure 2D graph and Supplemental Figure 1B).
Figure 2:
Hypoxia induces increased production of exosomes by TECs. (A) HIF-1α Western blot in MCT cellular extracts cultured under normoxic or hypoxic conditions. The bands are quantified and the HIF-1α and β -actin ratio is expressed in relative units (RUs). (B) Lactate quantification in culture medium from cells cultured under normoxic (N) and hypoxic (H) conditions. (C) Morphologic characterization of MCT exosomes by electronic microscopy. The size of exosomes is measured and expressed in nanometers. (D) Total exosome protein quantification (right graph). Protein extracts prepared from exosomes are normalized by protein extract volume per cell number and expressed in micrograms per 106 cells. CD63 Western blot analysis shows increased CD63 expression in hypoxia (left panel). Protein extracts prepared from exosomes are normalized by protein extract volume per cell culture number (micrograms per 106 cells; upper panel) or by concentration (10 µg; lower panel) and are assessed using antibody against exosomal protein marker CD63. (E) Western blot using CD63 and GFP antibodies of protein extract from MCT CD63-GFP cells using β -actin expression as loading control. (F) Confocal microscopy for GFP presence in MCT CD63-GFP cells cultured under normoxic or hypoxic conditions. Green signal represents the fusion protein CD63-GFP.
CD63 protein localizes within intraluminal bodies of cells, a site of exosome biogenesis before secretion, and it directly correlates with exosome production and quantity.21 , 22 Figure 2E ). After exposing CD63-GFP cells to hypoxic conditions for 48 hours, there was an increase in CD63-GFP protein (per cell) compared with CD63-GFP TECs under normoxic conditions (Figure 2F ). Collectively, these results indicate that hypoxic TECs secrete a higher number of exosomes compared with normoxic TECs.
We next investigated whether the exosomes secreted by hypoxic TECs carry TGF-β 1 mRNA to engage fibroblasts in the repair/fibrotic response. Using a Boyden chamber assay coated with basement membrane preparation, we show that GFP+ exosomes released from CD63-GFP TECs cross the basement membrane material and reach fibroblasts (Supplemental Figure 2). Next, equal amounts of exosomes extracted from normoxic and hypoxic cells were added to cultures of fibroblasts (murine embryonic-NIH-3T3 and renal tubulointerstitial-TFB). Exosomes from hypoxic TECs specifically induced proliferation, TGF-β 1 expression, α -SMA expression, F-actin expression, and type I collagen (α 1 chain) production upon 24 and 48 hours of coincubation, as assessed by quantitative RT-PCR and/or immunofluorescence (Figure 3, A–C ). Direct culture of fibroblasts in hypoxic conditions did not induce expression of genes associated with their activation including TGF-β 1 mRNA, suggesting that exosomes produced by hypoxic TECs specifically mediate this action rapidly in fibroblasts ( Figure 3, A–C and Supplemental Figure 3).
Figure 3:
Hypoxic TEC-derived exosomes lead to fibroblast activation. (A) MTT assay shows proliferation of 3T3 and TFB fibroblasts in normoxia (N), hypoxia (H24h), or exposed to exosomes extracted from MCT cells cultured under hypoxia for 48 hours (EMCT H48h). (B) TGF-β 1, α -SMA, and collagen-1 expression in normoxic (N) 3T3 and TFB fibroblasts that are treated with hypoxic exosomes secreted from MCT cells after 24 hours (EMCT H24h) and 48 hours (EMCT H48h) of hypoxia. Results are expressed in arbitrary units (AUs). (C) Immunofluorescence of α -SMA, collagen-1 (Col-1), and F-actin in 3T3 and TFB fibroblasts in normoxia (N), hypoxia (H24h), or treated with exosomes from MCT hypoxic cells (EMCT H48h). Fluorescence intensity per nuclei (FI/Nuclei) is quantified and expressed in relative units (RUs). *Significantly different from normoxic control cells (P <0.05).
Brefeldin A (BFA), a chemical reagent shown to inhibit exosome secretion from cells,23 β 1 mRNA content of 3T3 and TFB fibroblasts (Supplemental Figure 4, A and B). Next, hypoxic TECs were pretreated with actinomycin-D (ACT-D) to inhibit general transcription including that of TGF-β 1 mRNA and its subsequent accumulation in exosomes. An increase in TGF-β 1 mRNA was not observed in 3T3 and TFB fibroblasts incubated with exosomes from ACT-D pretreated cells (Supplemental Figure 4C). Cell death was similar in normoxic and hypoxic TEC cells at 24 and 48 hours of culture (Supplemental Figure 4D) and in TECs treated with Etoposide (0.6 mg/ml), a proapoptotic agent,24 β 1 mRNA levels remained unchanged (Supplemental Figure 4E).
Next, TGF-β 1 small interfering RNA (siRNA) was used to silence TGF-β 1 in TECs. In addition, TGF-β 1 siRNA was also used to silence target TGF-β 1 mRNA directly in exosomes (Figure 4A ). A significant decrease in TGF-β 1 mRNA expression was observed in cells with TGF-β 1 siRNA (Figure 4A ). Direct electroporation of exosomes with TGF-β 1 siRNA also significantly decreased the total amount of TGF-β 1 mRNA in the exosomes (Figure 4A ). When fibroblasts were treated with exosomes silenced for TGF-β 1, α -SMA expression, TGF-β 1 expression, and type I collagen expression remained unchanged, supporting the fact that fibroblast activation did not occur in this setting (Figure 4, A–C ). A possibility that TGF-β 1 mRNA targeting could occur due to direct transfer of TGF-β 1 siRNA from exosomes to fibroblasts was ruled out because expression of TGF-β 1 mRNA in the exosomes incubated with fibroblasts remained unchanged and did not decrease (Figure 4, A and B ). This was accomplished by using stoichiometrically limited amounts of siRNA in our transfections (Figure 4A ).
Figure 4:
Hypoxic TEC exosome-derived TGF-β 1 mRNA is functionally important for the activation of fibroblasts. (A) TGF-β 1 expression in MCT cells under different experimental conditions. In EMCT /H48h , MCT cells are exposed to hypoxia for 48 hours, the culture medium is collected, and exosomes are obtained for RNA extraction. In EMCT 48h + siRNA , MCT cells are exposed to hypoxia for 48 hours, and exosomes are extracted and treated with TGF-β 1 siRNA. In MCT+ siRNA/ EH48h , MCT cells are treated with siRNA, exposed to hypoxia for 48 hours, the culture medium is collected, and exosomes are obtained for RNA extraction; after this period, RNA is extracted for TGF-β 1 expression analyses. *P <0.05 compared with MCT cells under normoxia (MCT-N); + P <0.05 compared with EMCT H48h . (B) TGF-β 1, α -SMA, and collagen-1 expression and (C) immunofluorescence for α -SMA, collagen-1 (Col1a1), and F-actin in 3T3 and TFB normoxic (N) fibroblasts treated with hypoxic exosomes extracted from MCT cells under 48 hours of hypoxia (EMCT H48h). Exosomes were extracted from MCT cells cultured for 48 h under hypoxia and were then electroporated with TGF-β 1 siRNA (EMCT H48h + siRNA), and exosomes extracted from MCT cells treated with TGF-β 1 siRNA for 48 hours of hypoxia (MCT + siRNA/EH48h). Results are expressed in arbitrary units (AUs) for gene expression and the fluorescence intensity per nuclei (FI/Nuclei) is quantified and expressed in relative units (RUs) for immunofluorescence. *P <0.05.
Collectively, our study demonstrates that hypoxia-relevant fibrotic kidneys and hypoxic epithelial cells produce a significantly higher number of exosomes with specific genetic information that has the capacity to initiate proliferation and activation of fibroblasts. We demonstrate that exosomes released by the injured TECs can mediate fibroblast activation specifically by the transfer of TGF-β 1 mRNA.
Although exosomes are actively being studied in the context of cancer progression, their potential functional role in tissue repair/regeneration and fibrosis is completely unknown.1 , 24 – 26 β 1 is delivered to the fibroblasts by exosomes, and fibroblasts in turn translate it to initiate acute/rapid production of TGF-β 1 protein to facilitate proliferation and activation utilizing an autocrine-signaling loop.27 β 1 mRNA into exosomes that, in contrast to TGF-β 1 protein, can be translated to generate larger quantities of protein in a rapid fashion in cells that fuse with such exosomes. Such production of TGF-β 1 protein can initiate an autocrine process that ultimately leads to proliferation and activation of fibroblasts. This process is likely most efficient in early stages of injury before significant inflammation is encountered.
Concise Methods
Cell Culture
Mouse proximal tubular cells, NIH-3T3 mouse embryonic fibroblasts, and kidney interstitial fibroblasts (TFBs) were maintained in DMEM and proximal human TECs (HK-2) were maintained in DMEM/F12, cultured with 5% CO2 and 95% humidity, supplemented with FBS, centrifuged, and filtered in a 0.1-µm filter to remove exosomes contained in the FBS. Human or mouse TECs were divided into normoxia and hypoxia groups.
Exosome Extraction
Hypoxic conditions were created by flushing 5% CO2 and 95% N2 through a modified chamber. The culture system was sealed and incubated at 37°C for 24 or 48 hours. Exosomes were isolated. Briefly, collected culture medium from normoxia and hypoxia groups was centrifuged. The resultant supernatants were subjected to filtration on 0.1-µm pore filters, followed by ultracentrifugation. The resulting pellets were resuspended in culture medium for fibroblast treatment for 24 or 48 hours or suspended in PBS, pooled, and again ultracentrifuged at 100,000×g for 60 minutes. The final pellets of exosomes were resuspended in radioimmunoprecipitation assay buffer for protein extraction or Trizol for RNA extraction.
Kidneys Exosome Extraction
For kidney exosome extraction, 100 mg of kidney cortex was collected and submitted to mechanical and enzymatic digestion with collagenase and trypsin for 4 hours at 37°C. After this period, the sample was collected and centrifuged, and the supernatant was filtrated and ultracentrifuged as previously described for protein and RNA extraction.
Treatment of Fibroblasts with Exosomes
For fibroblast treatment, the exosome content of one of the flasks was analyzed using the microbicinchoninic acid assay and the reminiscent was used for treatment after being resuspended in culture media. Fibroblasts were treated with 30 µg/ml of exosomes for a period of 24 hours.
ACT-D and BFA Treatments
To deplete RNA from exosomes, MCT cells were treated with ACT-D (0.1 µg/ml; Sigma) or BFA (0.1 µg/ml; Sigma) and submitted to hypoxic conditions for 24 hours for exosome extraction and fibroblast treatment. In addition, in some experiments, 3T3 and TFB fibroblasts were treated with 1.0 µg/ml and 0.1 µg/ml of ACT-D for 24 hours, respectively. After this period, the culture medium was collected and exosomes were extracted. In both experiments, the concentration and exposition period were chosen based on the lowest dose showing biologic significance and low cell death.
Cell Viability Assays
MTT (3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide; Sigma) was used to detect cell viability and proliferation.
RNA Extraction and Quantitative PCR
Total RNA from exosome preparations and cells was extracted using Trizol, according to the manufacturer’s instructions. RT was done using a High Capacity cDNA Reverse Transcription Kit for real-time PCR. Primers for mouse TGF-β 1, α -SMA, collagen I, and human TGF-β were designed and synthesized by Eurofin Operon. QuantumRNA Universal 18S ribosomal RNA was used as endogenous control. Real-time analysis was performed using SYBR Green PCR Master Mix for real-time PCR.
Lactate Production
Lactate accumulation in the culture medium was determined using a colorimetric L-lactate assay kit from Eton Bioscience Inc, according to the manufacturer’s instructions.
Western Blotting
For Western blotting, aliquots of total protein extracts (10 μg) from cells or exosomes or the volume of exosome lysate corrected by the cell number were loaded and separated by 10% SDS-PAGE and the resulting proteins were then transferred onto a nitrocellulose membrane. After treatment with blocking solution, the membranes were incubated with rabbit polyclonal antibodies of CD63, rabbit polyclonal antibody for HIF-1α , or rabbit polyclonal antibody to actin. The membrane was then incubated with horseradish peroxidase–conjugated anti-rabbit or anti-mouse secondary antibodies and was examined by enhanced chemiluminescence. The membranes were then scanned, and the signal intensity of each band was quantified.
Immunofluorescence
For immunofluorescence, cells were grown on Labtek II glass slides and were fixed and permeabilized. After blocking with 5% BSA/PBS, FITC conjugated antibody to α -SMA, Cy5 conjugated antibody to phalloidin, and primary goat antibody to collagen I were added overnight at 4°C in 5% BSA/PBS. The FITC-labeled secondary anti-goat was added and cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI). Coverslips were mounted on glass slides with glycerin and analyzed using Nikon fluorescence microscopy.
TGF-β siRNA
RNAiFect reagent was used to deliver TGF-β pool siRNA or individual siRNA molecules to MCT cells. siRNA duplex oligonucleotides were transfected per the manufacturer's instruction. In brief, cells were plated the day before transfection, and were transfected and incubated for an additional 48 hours before harvesting. After this period, the culture medium was collected; exosomes were extracted for protein quantification and treatment of 3T3 and TFB fibroblasts. Exosomes at a total protein concentration of 150 µg/ml were electroporated with siRNA, pooled together for ultracentrifugation, and used to treat fibroblasts and/or RNA extraction.
Transfection of CD63-GFP
For constitutive expression of CD63 in MCT cells, transfection with pCT-CD63-GFP (SB) was followed by selection of stable clones with 1.5 μg/ml of puromycin.
TEM
The pelleted exosomes were mixed freshly prepared with paraformaldehyde/glutaraldehyde, fixed with osmium tetroxide, and dehydrated. Samples were embedded in resin. Ultrathin sections were cut with a diamond knife and sections were then placed on either copper or nickel mesh grids. The sections were stained with the heavy metal uranyl acetate for contrast and viewed on a Tecnai BioTwin.
UUO
Male C57BL6/6J mice were submitted to UUO and mice were euthanized on day 2 after surgery. The mice were injected with bromodeoxyuridine and hypoxyprobe, as previously described. The kidney cortex was extracted and weight for exosome extraction as described previously.
Immunohistochemistry
Masson’s trichrome staining was performed on paraffin-embedded tissue to detect collagen fibers according to standard protocol and the amount of collagen deposition was then digitally quantified. Formalin-fixed paraffin-embedded tissue sections were dehydrated and processed using MoM Kit (Vector Labs). Incubation of sections with primary hypoxyprobe, α -SMA antibodies was carried out overnight. Immunohistochemistry for bromodeoxyuridine was performed according to the manufacturer’s recommendations. Staining was quantified in renal sections of all mice in eight randomly chosen microscopic fields (cortex, inner medulla) at low magnification (×20).
Statistical Analyses
Data were expressed as mean ± SEM. Experimental and control groups were compared using the t test. Significance level for nullity hypothesis was set at 5% (P <0.05).
Disclosures
None.
This work was supported by grants from the National Institutes of Health (NIH) (CA125550, CA-155370, CA-151925, DK 081576, DK 055001), the Metastasis Research Center at the MD Anderson Cancer Center, and the Cancer Prevention and Research Institute of Texas. V.S.L. was funded by a NIH Research Training Grant in Gastroenterology at the Beth Israel Deaconess Medical Center (2T32DK007760-11). S.A.M. is a Human Frontiers Science Program fellow.
Published online ahead of print. Publication date available at www.jasn.org
See related editorial, “A New Look at Tubulointerstitial Communication with Exosomes,” on pages 330–332.
This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2012101031/-/DCSupplemental
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