Conceived and designed the experiments: GSDM PR MH SR DL. Performed the experiments: GSDM PR DK AG SR. Analyzed the data: GSDM PR MB RK SR DL. Contributed reagents/materials/analysis tools: PR MB RK BG WW HP MH DL. Wrote the paper: GSDM PR MH SR KL.
The authors have declared that no competing interests exist.
Kidney transplantation (RTx) leads to amelioration of endothelial function in patients with advanced renal failure. Endothelial progenitor cells (EPCs) may play a key role in this repair process. The aim of this study was to determine the impact of RTx and immunosuppressive therapy on the number of circulating EPCs.
We analyzed 52 RTx patients (58±13 years; 33 males, mean ± SD) and 16 age- and gender-matched subjects with normal kidney function (57±17; 10 males). RTx patients received a calcineurin inhibitor (CNI)-based (65%) or a CNI-free therapy (35%) and steroids. EPC number was determined by double positive staining for CD133/VEGFR2 and CD34/VEGFR2 by flow cytometry. Stromal cell-derived factor 1 alpha (SDF-1) levels were assessed by ELISA. Experimentally, to dissociate the impact of RTx from the impact of immunosuppressants, we used the 5/6 nephrectomy model. The animals were treated with a CNI-based or a CNI-free therapy, and EPCs (Sca+cKit+) and CD26+ cells were determined by flow cytometry.
Compared to controls, circulating number of CD34+/VEGFR2+ and CD133+/VEGFR2+ EPCs increased in RTx patients. There were no correlations between EPC levels and statin, erythropoietin or use of renin angiotensin system blockers in our study. Indeed, multivariate analysis showed that SDF-1 – a cytokine responsible for EPC mobilization – is independently associated with the EPC number. 5/6 rats presented decreased EPC counts in comparison to control animals. Immunosuppressive therapy was able to restore normal EPC values in 5/6 rats. These effects on EPC number were associated with reduced number of CD26+ cells, which might be related to consequent accumulation of SDF-1.
We conclude that kidney transplantation and its associated use of immunosuppressive drugs increases the number of circulating EPCs via the manipulation of the CD26/SDF-1 axis. Increased EPC count may be associated to endothelial repair and function in these patients.
Endothelial dysfunction is a typical finding in chronic kidney disease (CKD). It contributes to accelerated arteriosclerosis and impaired angiogenesis and, therefore, to high cardiovascular morbidity and mortality in these patients. However, after renal transplantation (RTx) endothelial function improves, even though substantial dysfunction is still observed in these patients
Interestingly, these vascular lesions can be repaired by i) migration and proliferation of endothelial cells contiguous to the lesions or by ii) the so-called endothelial progenitor cells (EPCs)
Patients with advanced renal failure were shown to have not only significant lower EPC numbers compared to healthy controls but, in addition, impaired EPC function
During the transformation process of EPCs into mature endothelial cells, human EPCs express different surface markers at distinct stages including CD133, CD34 and vascular endothelial growth factor receptor 2 (VEGF R2)
Immunosuppressive treatments of patients after RTx may directly affect the endothelial function
Clinical data of the study subjects are summarized in
Blood samples were obtained as part of a routine diagnostic or screening procedure. They were analyzed within 1 hour.
Circulating EPCs were identified by the expression of cell surface antigens, such as CD34+, CD133+, and VEGF-R2+. A) Density plot with forward (FSC) and side light scatter (SSC). P1-gate was selected for further analysis. B) Density plot of PE-conjugated anti-VEGF-R2 antibody versus FITC-conjugated anti-CD133 antibody. Cells double positively stained for VEGF-R2 and CD133 (quadrant Q2) represent CD133+ endothelial progenitor cells (CD133+/VEGFR2+ EPCs). C) Mouse-IgG1-FITC negative control and D) Mouse-IgG2a-PE negative control.
Cells sorted by FACS were further characterized by the expression of a specific endothelial cell marker or cultured in a human methylcellulose base media (A and B, respectively). A) CD133+/VEGFR2+ cells were immunohistochemically stained with an antibody against von Willebrand Factor (vWF). Negative control: omission of the primary antibody. B) Phenotypically, colonies formed by these cells in methylcellulose base media show the typical shape of early EPC-colonies with round immature cells in the center and dendritic or spindle cell-shaped peripheral cells (see magnification).
Circulating EPC – both, CD133+/VEGFR2+ and CD34+/VEGFR2+ EPCs – number is increased in RTx recipients when compared to controls (
EPC levels were directly quantified from whole blood taken from control subjects and patients (RTx) by flow cytometry, which identifies EPCs according to the expression of cell surface antigens, such as (A) CD133+ and VEGF-R2+; and (B) CD34+ and VEGF-R2+. P value compared to control group is indicated (Mann-Whitney test).
(A) CD133+ and VEGF-R2+; and (B) CD34+ and VEGF-R2+. CNI, calcineurin inhibitor. P value compared to control group is indicated (Mann-Whitney test).
Besides the immunosuppressive therapy, we analyzed if renal function (eGFR), diabetes mellitus and statin use interfere with the EPC count. In multivariate analysis we could not show any relation between eGFR or diabetes mellitus with circulating EPC number (
A) CD133+/VEGFR2+ EPCs; B) CD34+/VEGFR2+ EPCs.
CD133+EPC | ||
No stand. B | p-value | |
|
||
RTx (yes or no) | 0.157 | 0.760 |
eGFR (ml/min/1.73 m3) | −0.001 | 0.789 |
CNI (yes or no) | 0 037 | 0.908 |
CNI free (yes or no) | −0.037 | 0.898 |
Statin (yes or no) | 0.265 | 0.239 |
Antihypertensive therapy (yes or no) | 0.135 | 0.505 |
Diabetes mellitus (yes or no) | −0.119 | 0.644 |
SDF-1 alpha (pg/ml) | 0.001 | 0.020 |
|
||
|
0.001 | 0.000 |
Multivariate analysis. CD133+EPC was transformed to natural logarithm. B, no standardized regression coefficient beta. CNI, calcineurin inhibitor; eGFR, estimated glomerular filtration rate; SDF-1, stromal cell-derived factor 1 alpha; RTx, Kidney transplantation.
To investigate putative mechanisms in EPC mobilization, we measured plasma levels of SDF-1 (
To distinguish the impact of RTx from the impact of immunosuppressive drugs on the number of circulating EPCs - as well as to avoid potential confounders, such as concomitant diseases and medications present in human patients - we decided to use an additional experimental model. Since our RTx patients presented a 59 to 62% reduction in the GFR in comparison to controls (
Histological changes were examined by light microscopy in paraffin-embedded tissue with periodic acid-Schiff (PAS) (upper panels; magnification 40×) and picro Sirius red (lower panels; magnification 10×) stainings. Sham: sham animals; 5/6 Nx: nephrectomy; CNI: calcineurin inhibitor (cyclosporine A 5 mg/kg/day); CNI free: mycophenolate mofetil 30 mg/kg/day.
Sham | 5/6 Nx | |||
Vehicle | CNI | CNI free | ||
n = 8 | n = 6 | n = 5 | n = 5 | |
|
310±15 | 302±18 | 309±10 | 270±5 |
|
17±2 | 29±4 | 28±8 | 32±5 |
|
0.2±0.09 | 0.5±0.06 |
0.5±0.06 |
0.4±0.01 |
|
18±2 | 36±6 |
50±8 |
34±3 |
|
0.10±0.03 | 4.1±1.13 |
0.8±0.58# | 0.4±0.08# |
|
0.45±0.06 | 8.8±2.7 |
2.2±0.43# | 1.25±0.10# |
|
0.9±0.04 | 0.4±0.07 |
0.5±0.07 |
0.5±0.07 |
|
0.4±0.05 | 0.2±0.04 |
0.2±0.03 |
0.3±0.02 |
|
0.6±0.05 | 0.3±0.05 |
0.3±0.05 |
0.4±0.02 |
Results are mean ± SEM. BUN, blood urea nitrogen; Cr, creatinine, CrCl, creatinine clearance. Sham: sham animals. 5/6-nephrectomised rats (Nx) were treated (i.p) with: saline (vehicle); cyclosporine A 5 mg/kg/day (calcineurin inhibitior; CNI) and mycofenolat mofetil 30 mg/kg/day (CNI free). Results are mean ± SEM.
*P<0.05 compared to Sham.
Progenitor cells were defined by the surface expression of stem cell antigen-1 (Sca-1) and c-Kit antigens. This cell population represents highly immature cells that account for a small fraction of circulating mononuclear cells and include endothelial-committed precursors involved in compensatory angiogenesis at ischemic sites
Progenitor cells were defined by the surface expression of stem cell antigen-1 (Sca-1) and c-Kit antigens. The number of circulating progenitor cells (A) and CD26+ cells (B) was determined by flow cytometry 14 days after surgery/treatment. Sham: sham animals; 5/6 Nx: nephrectomy; CNI: calcineurin inhibitor (cyclosporine A 5 mg/kg/day); CNI free: mycophenolate mofetil 30 mg/kg/day. Results are mean ± SEM. *P<0.05 compared to Sham; #P<0.05 compared to vehicle.
Attenuation of the CD26 system can lead to increased concentration of SDF-1. Rats with renal failure and CNI treatment had lower circulating CD26+ cells number than sham and vehicle-treated 5/6 Nx rats. In CNI-free-treated rats the CD26+ cells number was slightly lower (
Few studies have yet reported on EPC counts in RTx (
EPC can be characterized by hematopoietic stem cell markers (clusters of differentiation) such as CD34 or CD133 combined with the expression analysis of an endothelial surface marker (VEGFR2 or KDR, von Willebrand factor, VE cadherin, CD146, CD31), uptake of Dil-acetylated lipoprotein, and lectin binding
To exclude influences of blood pressure, anti-hypertensive medication and other co-morbidities, we have chosen a control group of mostly hypertensive patients with normal kidney function for comparison to RTx recipients. Even though a higher mean blood pressure was associated with lower EPC counts in a study with RTx recipients
It has been shown that the graft function is an important determinant of EPC number and function in RTx recipients
Three factors could have influenced the EPC number in RTx recipients: the immunosuppressive therapy, RAS blockade and statin use. Indeed, we did not find a correlation between EPC levels and statin use or RAS blockade, drugs which have previously been reported to increase EPC numbers
To dissociate the impact of kidney transplantation itself and concomitant medication from the impact of immunosuppressive drugs (CNI vs. CNI-free) on the number of circulating EPCs, we have employed a defined animal model, the 5/6 nephrectomy to minimize confounders. 5/6 nephrectomy led to a decreased renal function (around 50% reduction in the GFR) which was comparable to the decreased renal function of RTx patients. 5/6 rats received no other drugs besides cyclosporine A or mycophenolate mofetil. Interestingly, as seen in uremic patients, vehicle-treated 5/6 Nx rats presented decreased circulating EPC counts, confirming that decreased renal function is directly associated to decreased EPC numbers. Even under this detrimental condition (uremia), CNI and CNI-free therapies improved the number of circulating progenitor cells in comparison to vehicle-treated 5/6 rats.
However, both immunosuppressants were not able to increase EPC number in sham-operated rats. These results suggest that, besides the immunosuppressive therapy, ischemic stress is also necessary to affect progenitor cell count in our model
EPCs participate in the repair of endothelial dysfunction
SDF-1 is constitutively expressed by most organs in the body. Interestingly, after kidney injury, its level is not only increased in the kidney, but also in the circulation
Finally, a strong evidence for the functional relevance of EPC for the positive effects of CNI/CNI-free on endothelial repair in the 5/6 model is the fact that treated rats presented reduced urinary albumin-to-creatinine and protein-to-creatinine ratios in comparison to vehicle-treated animals. It is well established that albuminuria/proteinuria reflects not only glomerular, but also generalized endothelial dysfunction, which explains its prognostic value (a sensitive marker) for renal and cardiovascular risks
In conclusion, we found that kidney transplantation and its associated use of immunosuppressive drugs lead to improved number of circulating EPCs. The nature and size of our study do not permit us to determine whether high levels of these cells can affect endothelial function in RTx cases. Rather, we can speculate that this increase in EPC count is associated with increased SDF-1 levels, suggesting increased endothelial repair and function in these patients.
Fifty two kidney transplant patients were included from the Transplantation Unit of the Department of Internal Medicine D, University Clinics Münster, Germany. As the most of the patients were hypertensive (70%), 16 age-matched subjects - of whom 11 with essential hypertension and normal kidney function - served as a control group to exclude implications of blood pressure as well as of antihypertensive treatment. Hypertension was controlled by medication in both groups.
EDTA-blood was obtained from all control subjects and patients. The blood samples of the patient cases were collected 50±46 and 77±62 months (mean ± SD) after kidney transplantation in both, CNI- and CNI free-groups, respectively.
The protocol was approved by the medical ethical committee of the University Clinics Münster (permit number 4IX Kosch-Lang). Written informed consent was obtained from all patients and control subjects.
The total number of circulating EPCs was analyzed by flow cytometry as previously described
After staining, cells were washed with PBS, lyzed with IO-Test 3 lyzing solution according to the manufacturer's instructions (Beckmann Coulter), and resuspended in PBS (1 ml). The double-labeled samples were then analyzed on a flow cytometer equipped with an electronic volumeter, which allows an exact measurement of the volume of the specimen aspired by the flow cytometer. A fixed volume of 200 µl was used, which, by the given dilution factor, allows the analysis of about 100.000 white blood cells in each single measurement. Thus the leukocyte, respectively the EPC, concentration was provided directly by the instrument (PAS III flow cytometer, equipped with a 20-mW 488-nm argon ion laser and 5 PMT's: FSC, SSC, FL1-3; Partec GmbH, Germany)
The threshold was set at the lower end of the forward scatter. Gates were set at forward scatter (FSC) and sideward scatter (SSC), including mononuclear cells and excluding PMNLs. Cells inside this gate were further analyzed with regard to their fluorescence properties. A gate was set around the region containing the double positively stained cells for the combinations: CD34-FITC/VEGF-R2-PE; CD34-FITC/CD133-PE; and CD34-FITC/CD45-PE. EPC number was determined by means of double positively stained mononuclear cells for VEGF-R2 and CD133 or VEGF-R2 and CD34 (CD133+/VEGFR2+ and CD34+/VEGFR2+ cells, respectively)
The reproducibility and variability of the method per patient over time had been previously determined by Rustemeyer et al. Moreover, our method presented high correlation with a cell culture method where the cytometrically purified stem cells (EPC) demonstrated their colony forming capacity
For further characterization cytospins of colonies were made. Cells were stained with 4′6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, Germany) and unconjugated monoclonal antibodies against von Willebrand Factor (vWF; Dako, Denmark). Immunodetection was visualized by FITC-labeled goat-anti-mouse-antibody (Dako, Denmark).
In addition, sorted CD133+/VEGFR2+ cells were directly transferred to a glass slide coated with poly-L-lysine (Sigma Aldrich, Germany), fixed with 4% paraformaldehyde and subsequently submitted to immunohistochemical analysis by using a polyclonal antibody against vWF (dilution 1∶100; Abbiotec, USA) and HRP-conjugated secondary antibody (dilution 1∶200; Vector laboratories, USA). Omission of the primary antibody was used as negative control.
CD26 and stromal-derived factor 1 alpha (SDF-1) levels were measured in patients' and controls' plasma by using commercial ELISA kits (human DPPIV/CD26 and human CXCL12/SDF-1 alpha immunoassay, respectively, R&D Systems). Samples for CD26 determination were 100-fold diluted in Calibrator Diluent according to manufacturer's specifications, while SDF-1 determination does not require dilution. In both assays, the antibodies were raised against the human recombinant factors.
Renal disease was induced in Sprague Dawley rats by 5/6-resection of renal tissue as previously described
Circulating stem/progenitor cells and CD26+ cells were analyzed by flow cytometry as previously described
Analyses were performed with the PASW, Version 18.0 (SPSS Inc., Chicago, IL). Non-normal data are presented as median and interquartil range; data found to be normally distributed are presented as means ± SD. The Mann-Whitney test and Kruskal-Wallis test were used to compare two or all three groups, respectively. Variables based on proportions were analyzed by chi-square test. Multivariate regression analyses were performed to assess associations between CD133+EPC number and other parameters with regards to potentially confounding factors. Results are described as regression coefficient Beta (Stand. B). The two-sided p<0.05 was considered to reflect statistical significance.
Experimental data is presented as mean ± SEM. Comparison among groups was performed by Kruskal-Wallis test. A level of P<0.05 was accepted as statistically significant. Analyses were performed using GraphPad Prism version 4.0.
Concentration of plasma CD26 (A) and stromal cell-derived factor 1 alpha (SDF-1) (B) in control and renal transplant patients according to their immunosuppressive therapy regimen. CNI: calcineurin inhibitor. The clinical characteristics of this specific control and patient population are given in
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Clinical characteristics of kidney transplant patients.
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Available studies on endothelial progenitor cells (EPCs) after kidney transplantation (RTx).
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Clinical characteristics of control and Kidney transplant patients related in
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The authors thank Professor A. Jacobi, Medizinische Klinik und Poliklinik D, UK Münster, for FACS facilities placed at her laboratory and for her technical advices. The authors also thank Katrin Beul and Petra Haussmann for excellent technical assistance.