TY - JOUR
AU - Segal, Mark, S.
AB - calcineurin inhibitor, circulating endothelial progenitor cell, endothelial dysfunction, mitomycin Introduction Thrombotic microangiopathies (TMA) are multisystem disorders characterized by the formation of platelet-rich, fibrin-poor thrombi within the microvasculature. While there have been major advances into the cause of certain types of TMAs that have led to rational treatment choices [1], TMA secondary to chemotherapeutic and calcineurin inhibitors remains a black box both in terms of pathogenesis and treatment. Here we report a case of mitomycin-associated haemolytic uremic syndrome (HUS) resistant to conventional treatment but which responded to chronic end-stage renal disease management consisting of haemodialysis and erythropoietin treatment. We hypothesized that haemodialysis and erythropoietin may play a role in ‘healing’ the endothelial injury. To support our hypothesis, we measured the markers of endothelial cell injury (circulating endothelial cells) and a marker of endothelial repair (CD34+ endothelial progenitor cell migratory activity) in a second case of TMA secondary to calcineurin therapy. We demonstrate that, in drug-induced HUS, there is increased endothelial injury and a defective repair mechanism and that correcting uraemia, with renal replacement therapy, and treatment with specific medications aimed at enhancing endothelial repair may be able to alter the course of the disease. These findings not only suggest a mechanism for drug-induced TMA, but also suggest a rational line of therapy for a disease that heretofore was treated with supportive care. Methods Enumeration of circulating endothelial cells Strict adherence to the Declaration of Helsinki was followed in this study. The study protocol was approved by the Institutional Review Board at the University of Florida and written informed consent was obtained prior to study. Circulating endothelial cells (CECs) were isolated from peripheral blood mononuclear cells (PBMCs) using immunomagnetic beads (Dynabeads M-500, Dynal Biotech Inc., Oslo, Norway) conjugated with P1H12 (Chemicon, Temecula, CA), a murine, monoclonal antibody specific for human endothelial cells. Quantitation of CEC was performed by identification of the cells using a Zeiss Axiophot microscope (Carl Zeiss Inc., Thornwood, NY). Consistency of conjugated immunomagnetic beads were confirmed with testing on a standard dilution of fixed human umbilical vein endothelial cells. Isolation of CD34+ endothelial progenitor cells (EPCs) Blood was collected by routine venipuncture into CPT™ Tubes with heparin (BD Biosciences, Franklin Lakes, NJ). The CD34+ cells were isolated from PBMCs, with magnetic microbeads conjugated with an anti-CD34 antibody (Miltenyi Biotec Inc., Auburn, CA) using an automated magnetic selection autoMACS™ (Miltenyi Biotec Inc.). The selected cells were confirmed to be CD34+ cells by fluoresence-activated cell-sorting (FACS) analysis after co-staining with phycoerythrin (PE)-conjugated anti-CD34 (Miltenyi Biotec Inc.) and fluorescein isothiocyanate (FITC)-conjugated anti-CD45 and FITC-conjugated anti-CD34 and PE-conjugated anti-VEGFR2. Purification yielded a population of cells 95% of which were CD34+. Stromal-derived-factor-1-induced chemotaxis A polycarbonate membrane (8 µm pores) (Neuro Probe, Gaithersburg, MD) coated with 10% bovine collagen was placed on the bottom of a Boyden Chamber (Neuro Probe, Gaithersburg, MD) loaded with increasing concentrations of SDF-1 (R&D Systems Inc., Minneapolis, MN). EPCs stained with Calcein-AM (Molecular Probes) were loaded in the top chamber. After 4.5 h at 5% CO2 at 37°C, the relative fluorescence, determined with the Synergy™ HT (Bio-Tek Instruments, Inc., Winooski, VT) with an excitation of 485 ± 20 and an emission of 528 ± 20, of the lower chamber media determined the percentage of migrating cells. Detection of nitric oxide produced by EPC CD34+ EPCs were incubated with 5 µM 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) diacetate (Invitrogen, Carlsbad, CA) for 30 min at 37°C in the dark. The fluorescence of DAF-FM increases by about 160-fold when it reacts with NO. CD34+ EPCs were placed on glass bottom microwells (MatTek Corp., Ashland, MA) for imaging in a confocal microscope at excitation and emission maxima at 495 and 515 nm, respectively. Intensity of fluorescence was quantified using LSM 510 (version 3.0 SP3) software for the Carl Zeiss Laser Scanning Microscope and expressed as fluorescence per cell. Case 1 Mr X is a 20-year-old Caucasian man who presented with fatigue of a few weeks duration. Six months earlier he was diagnosed with poorly differentiated gastric adenocarcinoma, which was treated with surgical resection and mitomycin at a dose of 12 mg/m2/month for 4 months (cumulative dose 48 mg/m2). Upon admission he was found to be anaemic, thrombocytopenic, hypertensive and azotemic with non-oliguric acute renal failure. Other laboratory findings included: haematocrit 22.7%, lactic dehydrogenase (LDH) 1074 U/l, haptoglobin <10 mg/dl (<0.1 g/l), platelets 17 000/mm3 (17 × 109/l). Urinalysis revealed 1+ albuminuria and haematuria (10–20 RBCs/hpf) with rare granular casts on microscopic examination of urinary sediment. Plasmapheresis was initiated but required immediate discontinuation due to the development of acute pulmonary oedema requiring endotracheal intubation and mechanical ventilation. Echocardiography revealed an ejection fraction of 50%, and the episode was attributed to acute lung injury associated with administration of pooled plasma. The patient responded well to diuresis and was extubated within 72 h. During this time his creatinine had plateaued at 2.3 mg/dl (203 µmol/l), while his haematocrit and platelet counts continued to decline with persistent elevation of LDH levels. Six consecutive treatments with staphylococcal protein-A immunoadsorption were performed over a period of 23 days. His haematocrit stabilized at 28%, platelet counts remained in the 40–50 000 range, renal function remained stable with serum creatinine of 2.3 mg/dl (203 µmol/l) and creatinine clearance was calculated at 24 ml/min (0.40 ml/s). Twelve days after discharge the patient was re-admitted with seizures and pulmonary oedema requiring mechanical ventilation. Renal function was found to be severely impaired with a BUN of 111 mg/dl (39.6 mmol/l) and creatinine of 4.8 mg/dl (424 µmol/l). His haematocrit and platelet counts were 21 and 19 000, respectively, with a LDH of 2890 U/l. MRI scanning of the head did not reveal any focal neurological abnormalities. Renal replacement therapy with conventional haemodialysis [4 h three times weekly using a F80 polysulfone dialysis filters (Fresenius Medical Care, Walnut Creek, CA), bicarbonate dialysate, and heparin sodium as standard anticoagulant] and erythropoietin (erythropoietin-α 10 000 IU three times a week) was initiated. After receiving two treatments, he was extubated and improvement in blood counts was noted. He was discharged with a haematocrit and platelet counts of 30 and 92, respectively, to continue out-patient haemodialysis. After 4 weeks of treatment his haematocrit was 39.5 and platelets had risen to 281 000, with LDH in the normal range. Residual renal function was measured and his creatinine clearance was 17 ml/min (0.28 ml/s). Dialysis was discontinued and after 4 months, he has no signs of ongoing HUS with his creatinine stable at 1.7 mg/dl (150 µmol/l) and a creatinine clearance of 30 ml/min (0.50 ml/s). Case 2 Ms Y is a 29-year-old White female with pre-B-cell acute lymphoblastic leukaemia initially diagnosed in August 2004. The patient underwent induction chemotherapy with hyper-CVAD (fractionated cyclophosphamide, vincristine, adriamycin and dexamethasone) regimen. In all she received eight cycles of hyper-CVAD, four of cycle A and four of cycle B. Then, she began maintenance chemotherapy with 6-mercaptopurine, methotrexate, prednisone and vincristine. Later, she was diagnosed with relapsed acute lymphoblastic leukaemia and given re-induction chemotherapy. She received allogeneic peripheral blood stem cell transplant, and her post-transplant course was complicated with veno-occlusive disease, congestive heart failure, respiratory failure and sepsis. During her post-transplant period, she was placed on Prograft as one of her immunosuppression medications. Her baseline creatinine was 0.6–0.7 mg/dl (53–62 µmol/l) pre-operatively, but post-operatively she had gradual decline in her renal function with a creatinine increase to 2.7 mg/dl (239 µmol/l) with oliguria. In addition, she had pancytopenia as well as fever. Her LDH was elevated at 1200 U/l and her peripheral smear showed schistocytes. A diagnosis of possible calcineurin inhibitor-induced HUS-TTP was made. Prograft was discontinued and fresh frozen plasma was given to correct her coagulopathy, but she never received plasma exchange. Because of her acute renal failure and volume overload, she was initiated on renal replacement therapy and erythropoietin (Darbepoetin alfa 100 mcg a week). She received 10 conventional haemodialysis [4 h each using a F160 polysulphone dialysis filter (Fresenius Medical Care, Walnut Creek, CA), and bicarbonate dialysate] treatments over 13 days and started showing signs of renal recovery. Haemodialysis was stopped and she recovered her renal function completely with her creatinine returning to her baseline of 0.7 mg/dl (62 μmol/l). Special studies in Case 2 Endothelial injury can be monitored via enumeration of CECs [2,3]. At the time of diagnosis Ms Y's CEC cell number was 82 cells/ml (normal level 19 ± 7 cells/ml). When haemodialysis was being discontinued, her CEC cell number improved to 46 cells/ml (Table 1). Table 1. Special studies and platelet levels in Case 2 Time of analysis . CECa (cells/ml) . EPC migration to 100 nM SDF-1b . Intracellular NO bioavailabilityc . Plateletsd per mm3 . Day 1 82 0 878 ± 101 28 000 Day 5 ND 4 ± 1.4 1961 ± 88 40 000 Day 13 46 11.7 ± 0.2 1849.7 ± 91 28 000 Time of analysis . CECa (cells/ml) . EPC migration to 100 nM SDF-1b . Intracellular NO bioavailabilityc . Plateletsd per mm3 . Day 1 82 0 878 ± 101 28 000 Day 5 ND 4 ± 1.4 1961 ± 88 40 000 Day 13 46 11.7 ± 0.2 1849.7 ± 91 28 000 aCEC number in healthy individuals is 19 ± 7 and for patients on haemodialysis 29 ± 18 [11]. bEPC migration is expressed as percentage increase of cells migrating to 100 nM SDF-1 as opposed to 0 nM SDF-1. In healthy controls there is a 59 ± 14% increase, while in patients on haemodialysis the increase is only 27 ± 8% [4]. cLevel of NO of healthy controls is 1400 ± 377.7 AU of DAF-FM fluorescence per cell and for patients on haemodialysis 400 ± 86 Arbitrary Units (AU) per cell. dTo convert to platelets/l multiply by 106. Open in new tab Table 1. Special studies and platelet levels in Case 2 Time of analysis . CECa (cells/ml) . EPC migration to 100 nM SDF-1b . Intracellular NO bioavailabilityc . Plateletsd per mm3 . Day 1 82 0 878 ± 101 28 000 Day 5 ND 4 ± 1.4 1961 ± 88 40 000 Day 13 46 11.7 ± 0.2 1849.7 ± 91 28 000 Time of analysis . CECa (cells/ml) . EPC migration to 100 nM SDF-1b . Intracellular NO bioavailabilityc . Plateletsd per mm3 . Day 1 82 0 878 ± 101 28 000 Day 5 ND 4 ± 1.4 1961 ± 88 40 000 Day 13 46 11.7 ± 0.2 1849.7 ± 91 28 000 aCEC number in healthy individuals is 19 ± 7 and for patients on haemodialysis 29 ± 18 [11]. bEPC migration is expressed as percentage increase of cells migrating to 100 nM SDF-1 as opposed to 0 nM SDF-1. In healthy controls there is a 59 ± 14% increase, while in patients on haemodialysis the increase is only 27 ± 8% [4]. cLevel of NO of healthy controls is 1400 ± 377.7 AU of DAF-FM fluorescence per cell and for patients on haemodialysis 400 ± 86 Arbitrary Units (AU) per cell. dTo convert to platelets/l multiply by 106. Open in new tab While CECs are thought to be a marker of endothelial injury, EPCs are thought to reflect inherent endothelial repair ability. CD34+ EPC migration and bioavailable NO are measures of CD34+ EPC health [4]. At the time of diagnosis, Ms Y's CD34+ EPCs were defective in their ability to migrate and had a low level of bioavailable NO (Figure 1; Table 1). These parameters both improved by day 13. Fig. 1. Open in new tabDownload slide Bioavailable NO increases with institution of haemodialysis and erythropoietin therapy. EPCs were incubated with DAF-FM and imaged with a confocal microscope (magnification: ×200). Fig. 1. Open in new tabDownload slide Bioavailable NO increases with institution of haemodialysis and erythropoietin therapy. EPCs were incubated with DAF-FM and imaged with a confocal microscope (magnification: ×200). Discussion Several important findings can be appreciated from the cases presented. First, in both cases, the HUS seemed to respond to the institution of haemodialysis and erythropoietin therapy. Of course, the response could be to the cessation of the offending agent. But in the case of Mr X, the mitomycin was remote relative to the diagnosis of HUS. Second, consistent with the idea that drug-induced HUS is a disease of the endothelium, the CEC number was increased by 4-fold in Ms Y at the time of diagnosis and decreased as the disease seemed to improve. CECs have previously been demonstrated to be markedly increased in conditions associated with endothelial cell injury, such as myocardial infarction [5] and thrombotic thrombocytopenic purpura [6]. The third important finding is that CD34+ EPC function was perturbed. The term EPC not only refers to colonies grown from PBMCs or from purified populations of CD34+ or CD133+ haematopoietic cells [7] but also to the CD34+ or CD133+ cells in the peripheral circulation. After the first case, we hypothesized that uraemia itself may have detrimental effects on CD34+ EPC function. Thus, during the second case we measured CD34+ EPC function. We found that CD34+ EPC function started to improve with the initiation of haemodialysis and erythropoietin therapy prior to the patient's condition improving (i.e. when the platelet count was still depressed and the patient was dialysis dependent). Previously, we have demonstrated that patients on chronic haemodialysis secondary to diabetes have poor CD34+ EPC migration, even after haemodialysis, that is only enhanced with incubation with a NO donor. This is consistent with previous reports that uraemia appears to be suppressive of EPC function [8], and erythropoietin seems to improve EPC function [9]. As with the CEC number, improvement in CD34+ EPC function was detected prior to clinical improvement. The observation that CD34+ EPC migration improved as the patient's condition improves is not unexpected, since one would expect EPC function to normalize as the patient's health improved. The mechanistic insight is that there are treatments that stabilize endothelial function and/or enhance EPC migration that may hasten the improvement in drug-induced HUS. In addition to haemodialysis for uremic patients and erythropoietin, HMG CoA reductase inhibitors have also been shown to increase EPC numbers and their ability to migrate and form colonies [7]. HMG Co-A inhibitors have the added benefit of stabilizing vascular endothelial cells by a number of mechanisms including decreased expression of VCAM, decreased expression of E-selectins, as well as promoting angiogenesis by enhancing Akt phosphorylation [10]. Thus, haemodialysis, erythropoietin and HMG CoA inhibitors may be particularly effective in drug-induced TMA by limiting injury to endothelial cells and increasing the body's ability to carry out endothelial repair via EPC activity. In conclusion, these cases demonstrate that drug-induced HUS is likely due to endothelial injury and that EPC function may not only be a target of treatment but may also be used to monitor the state of the disease. If the health of the vascular endothelium is thought of as a balance between endothelial injury and endothelial repair, in drug-induced HUS the balance is tipped, with endothelial injury, as determined by CECs, outweighing the body's ability to carry out endothelial repair. In drug-induced HUS, treatments aimed at stabilizing, the vascular endothelium, and increasing the number and function of EPCs, to carry out repair may improve the disease course. Acknowledgements This work was supported by National Institutes of Health Grant DK02537, Gatorade Research funds, and Baxter Renal Discovery Grant to M.S.S. We thank Dr R. J. Johnson for critical reading of the manuscript. Conflict of interest statement. None declared. References 1 Murrin RJA , Murray JA . 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Endothelial progenitor cell proliferation and differentiation is regulated by erythropoietin - Rapid Communication , Kidney Int , 2003 , vol. 64 (pg. 1648 - 1652 ) Google Scholar Crossref Search ADS PubMed WorldCat 10 Kureishi Y , Luo Z , Shiojima I , et al. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals , Nat Med , 2000 , vol. 6 (pg. 1004 - 1010 ) Google Scholar Crossref Search ADS PubMed WorldCat 11 Koç M , Bihorac A , Segal M . Circulating endothelial cells as potential markers of the state of the endothelium in hemodialysis patients , Am J Kidney Dis , 2003 , vol. 42 (pg. 704 - 712 ) Google Scholar Crossref Search ADS PubMed WorldCat © The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
TI - Mitomycin- and calcineurin-associated HUS, endothelial dysfunction and endothelial repair: a new paradigm for the puzzle?
JF - Nephrology Dialysis Transplantation
DO - 10.1093/ndt/gfl586
DA - 2007-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/mitomycin-and-calcineurin-associated-hus-endothelial-dysfunction-and-jme83JLUT5
SP - 617
EP - 620
VL - 22
IS - 2
DP - DeepDyve
ER -