Urinary CD133⁺ Extracellular Vesicles Are Decreased in Kidney Transplanted Patients with Slow Graft Function and Vascular Damage

<div><p>Extracellular vesicles (EVs) present in the urine are mainly released from cells of the nephron and can therefore provide information on kidney function. We here evaluated the presence of vesicles expressing the progenitor marker CD133 in the urine of normal subjects and of patients undergoing renal transplant. We found that EV expressing CD133 were present in the urine of normal subjects, but not of patients with end stage renal disease. The first day after transplant, urinary CD133⁺ EVs were present at low levels, to increase thereafter (at day 7). Urinary CD133⁺ EVs significantly increased in patients with slow graft function in respect to those with early graft function. In patients with a severe pre-transplant vascular damage of the graft, CD133⁺ EVs did not increase at day 7. At variance, the levels of EVs expressing the renal exosomal marker CD24 did not vary in the urine of patients with end stage renal disease or in transplanted patients in respect to controls. Sorted CD133⁺ EVs were found to express glomerular and proximal tubular markers. These data indicate that urinary CD133⁺ EVs are continuously released during the homeostatic turnover of the nephron and may provide information on its function or regenerative potential.</p></div

Median values of CD24⁺ and CD133⁺ EV levels in the urine of transplanted patients at day 1 and 7 after transplant (D1 and D7), divided according to the graft vascular lesions (VS), as described in Material and methods.

<p>No increase in urinary CD133⁺ EVs was observed in VS2 patients. Statistical analysis was performed as described in the Materials and methods section. * = p<0.05.</p

Characteristics of the transplanted patients, ESRD patients and normal subjects in the study.

<p><b>ECD:</b> expanded criteria donors according to Crystal City Meeting criteria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104490#pone.0104490-Rosengard1" target="_blank">[35]</a>. <b>GFR:</b> glomerular filtration rate. <b>Histological score:</b> performed on pretransplant biopsies according to a semiquantitative method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104490#pone.0104490-Remuzzi1" target="_blank">[25]</a>. <b>APKD:</b> adult polycystic kidney disease; <b>Gn:</b> glomerulonephritis; <b>LD:</b> living donor; <b>DD:</b> deceased donor. <b>ID:</b> immunosuppressive therapy. <b>ESRD:</b> end-stage kidney disease. Continuous variables are express as mean ± SD.</p

Detection of CD133⁺ EVs in transplanted patients.

<p>Panel A and B. Representative dot plots showing the gating strategy of latex bead-conjugated EVs (A) and the fluorescence detection of isotype or CD133 on EVs from a transplanted patients at day 1 or 7.</p

CD24⁺ and CD133⁺ EVs in the urine of normal subjects, ESRD and transplanted patients.

<p>Panel A. Levels of urinary CD24⁺ and CD133⁺ EVs in normal subjects (H), ESRD patients with residual diuresis and transplanted patients at day 1, 7 or 30 after transplant (D1, D7 and D30, respectively). A significant reduction of CD133⁺ EVs but not of CD24⁺ EVs was observed in ESRD and transplanted patients at day 1. Data are expressed as % positive beads and are mean ± SD of all samples. <b>Panel B and C</b>. Representative cytofluorimetric histograms (B) and median values (C) of CD24⁺ and CD133⁺ EV levels in the urine of patients with EGF (n = 13) and SGF (n = 12) at day 1 and 7 after transplant. Urinary CD133⁺ EVs but not CD24⁺ EVs were lower in EGF patients at day 1, and significantly increased at day 7. Statistical analysis was performed as described in the Materials and methods section. * = p<0.05.</p

Characterization of urinary EVs in transplanted patients.

<p>Urinary EVs were obtained from patients (n = 25) undergoing renal transplant. <b>Panel A</b>. Mean number of EVs/day in the urine of transplanted patients were similar at day 1 and 7 after transplant, counted as described in Material and Methods. Data are mean ± SD of all samples. <b>Panel B and C</b>. Characterization of EVs showing expression of CD9 and CD81 exosome markers and NanoSight analysis of size distribution profile. Similar results were obtained for samples from all transplanted patients at day 1 and 7. <b>Panel D</b>. Representative western blot analysis showing the donor origin of the extracellular vesicle using typing sera containing antibodies directed against HLA class I antigens of the donor not shared by the recipient, at 1 and 7 days after transplant. In the table are reported the HLA typing of the recipient and the donor and the HLA-antigen recognized by the typing serum (TORP1262). HLA-B5 (bold) is present in the donor and not in the recipient. Three patients were tested with similar results.</p

Additional file 1: Figure S1. of The effects of glomerular and tubular renal progenitors and derived extracellular vesicles on recovery from acute kidney injury

Renal cell proliferation in IRI-mice treated with Gl-MSCs-derived EVs. (A) Quantification of BrdU-positive cells/high power field (HPF) was performed in renal sections of IRI mice injected with vehicle alone (IRI-CTL), 400 × 106 EVs produced by Gl-MSCs (IRI-Gl-MSC-EV), 400 × 106 EVs produced by Gl-MSCs and obtained by floating process (IRI-Gl-MSC-EV-float), 400 × 106 EVs produced by Gl-MSCs and treated with RNase (IRI-RNase-Gl-MSC-EV), and in sham-operated SCID mice. ANOVA with Dunnett’s multiple comparison test was performed, (* p <0.05). (B) Representative micrographs of BrdU staining preformed on section of kidneys 2 days after IRI and treatments injection. Original magnification: ×40. (DOCX 135 kb

Neutrophil Gelatinase Associated Lipocalin Is an Early and Accurate Biomarker of Graft Function and Tissue Regeneration in Kidney Transplantation from Extended Criteria Donors

<div><p>Background</p><p>Delayed graft function (DGF) is an early complication of kidney transplantation (KT) associated with increased risk of early loss of graft function. DGF increases using kidneys from extended criteria donors (ECD). NGAL is a 25KDa protein proposed as biomarker of acute kidney injury. The aim of this study was to investigate the role of NGAL as an early and accurate indicator of DGF and Tacrolimus (Tac) toxicity and as a mediator of tissue regeneration in KT from ECD.</p><p>Methods</p><p>We evaluated plasma levels of NGAL in 50 KT patients from ECD in the first 4 days after surgery or after Tac introduction.</p><p>Results</p><p>Plasma levels of NGAL at day 1 were significantly higher in DGF group. In the non DGF group, NGAL discriminated between slow or immediate graft function and decreased more rapidly than serum creatinine. NGAL increased after Tac introduction, suggesting a role as marker of drug toxicity. <i>In vitro</i>, hypoxia and Tac induced NGAL release from tubular epithelial cells (TEC) favoring an autocrine loop that sustains proliferation and inhibits apoptosis (decrease of caspases and Bax/Bcl-2 ratio).</p><p>Conclusions</p><p>NGAL is an early and accurate biomarker of graft function in KT from ECD favoring TEC regeneration after ischemic and nephrotoxic injury.</p></div

Evaluation of serum creatinine (sCr in A) and plasma NGAL (pNGAL in B) in the first 4 consecutive days after KT in non-DGF patients (n = 36).

<p>NGAL significantly decreased starting from day 2 to day 4 after KT reaching a value near to normal levels (100 ng/ml). Each value of pNGAL was significantly lower in comparison to the previous day (*p<0.05). By contrast, sCr remained stable or even increased (p>0.05).</p

Evaluation of plasma NGAL levels at day 1 after KT in patients subdivided on the basis of graft function (DGF: Delayed Graft Function; SGF: Slow Graft Function; IGF: Immediate Graft Function).

<p>(A) NGAL was significantly higher in DGF (662.7 ±97.2 ng/ml, n = 14) vs. non-DGF patients (SGF + IGF: 379.7±139.7 ng/ml, n = 36; #p<0.00001). A statistical significance was also observed comparing DGF vs SGF (444.1±149.5 ng/ml, n = 19; *p<0.001) or IGF (307.8±101.8 ng/ml, n = 17; $p<0.00001) and SGF vs IGF (§p<0.01). NGAL levels of KT from living donors (LD) were used as control. (B) The Area Under the ROC curve (AUC) of NGAL as biomarker of DGF at day 1 post-KT was 0.94.</p