SEARCH

SEARCH BY CITATION

Keywords:

  • Endothelial dysfunction;
  • microvascular injury;
  • simultaneous pancreas–kidney transplantation;
  • type 1 diabetes mellitus

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. Disclosure
  10. References

Simultaneous pancreas–kidney transplantation (SPK) is an advanced treatment option for type 1 diabetes mellitus (DM) patients with microvascular disease including nephropathy. Sidestreamdarkfield (SDF) imaging has emerged as a noninvasive tool to visualize the human microcirculation. This study assessed the effect of SPK in diabetic nephropathy (DN) patients on microvascular alterations using SDF and correlated this with markers for endothelial dysfunction. Microvascular morphology was visualized using SDF of the oral mucosa in DN (n = 26) and SPK patients (n = 38), healthy controls (n = 20), DM1 patients (n = 15, DM ≥ 40 mL/min) and DN patients with a kidney transplant (KTx, n = 15). Furthermore, 21 DN patients were studied longitudinally up to 12 months after SPK. Circulating levels of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and soluble thrombomodulin (sTM) were measured using ELISA. Capillary tortuosity in the DN (1.83 ± 0.42) and DM ≥ 40 mL/min (1.55 ± 0.1) group was increased and showed reversal after SPK (1.31 ± 0.3, p < 0.001), but not after KTx (1.64 ± 0.1). sTM levels were increased in DN patients and reduced in SPK and KTx recipients (p < 0.05), while the Ang-2/Ang-1 ratio was normalized after SPK and not after KTx alone (from 0.16 ± 0.04 to 0.08 ± 0.02, p < 0.05). Interestingly, in the longitudinal study, reversal of capillary tortuosity and decrease in Ang-2/Ang-1 ratio and sTM was observed within 12 months after SPK. SPK is effective in reversing the systemic microvascular structural abnormalities in DN patients in the first year after transplantation.


Abbreviations
Ang-1

angiopoietin-1

Ang-2

angiopoietin-2

AUC

area under the curve

BMI

body mass index

CAD

coronary artery disease

DM

diabetes mellitus type 1

DN

diabetic nephropathy

ELISA

enzyme-linked immuno sorbent assay

eGFR

estimated glomerular filtration rate

KTx

kidney transplantation

MDRD

modification of diet in renal disease

MMF

mycophenolatemofetil

SDF

side-streamdarkfield

SEM

standard error of the mean

SPK

simultaneous pancreas–kidney transplantation

SPKFU

simultaneous pancreas–kidney transplantation follow-up

sTM

soluble thrombomodulin

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. Disclosure
  10. References

Diabetes mellitus (DM) is a severe metabolic disease that results in macrovascular as well as microvascular complications including retinopathy, nephropathy and neuropathy [1, 2]. The majority of these microvascular complications is associated with endothelial dysfunction, and upregulation of different angiogenic growth factors including angiopoietin-2 (Ang-2) [2, 3] and soluble thrombomodulin (sTM) release in the circulation [4, 5]. Negative interference of Ang-2 with Ang-1 mediated Tie-2 signaling results in disruption of perivascular stromal cell–endothelial cell interaction, subsequent vessel destabilization and abnormal microvascular remodeling [6, 7].

Simultanous pancreas–kidney transplantation (SPK) has become an important option in the treatment of diabetic patients with nephropathy and seems to be superior to kidney transplantation alone (KTx), since SPK is associated with a better glycemic control as well as improved patient survival [8-10]. It has been shown that SPK and pancreas transplantation alone effectively prevent the recurrence of diabetic nephropathy (DN) [11-15]. However, in terms of stabilization or improvement of preexisting neuropathy and retinopathy, debate still continues on the benefit of SPK [16-20]. Early noninvasive monitoring of the microvasculature may be of clinical value to assess progression of microvascular disease during diabetes and treatment efficacy after SPK. Sidestreamdarkfield (SDF) imaging has recently emerged as a noninvasive tool to visualize the human microcirculation and to assess microvascular remodeling [21]. We have previously used this validated technique to compare the labial mucosal capillary tortuosity, as markers for microvascular disease, in diabetic patients with and without coronary artery disease (CAD) and healthy nondiabetic controls. Diabetic patients showed increased capillary tortuosity, especially in patients with CAD [22]. However, to our knowledge no previous studies have used SDF imaging to monitor microvascular alterations before and after SPK.

In this study, the effects of SPK on microvascular damage is assessed in a cross-sectional and longitudinal study using SDF imaging and correlated with markers for endothelial dysfunction. We hypothesized that SPK will reverse the microvascular damage in patients with DN.

Material and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. Disclosure
  10. References

Patients

All procedures were approved by the institutions Medical Ethical Committee. Written informed consent has been obtained from all the patients and healthy controls. A total of 114 persons (64 males and 50 females) were enrolled in this cross-sectional study after giving informed consent. Thirty-eight patients with simultaneous SPK were included (SPK group). Furthermore, we included 15 patients with a functioning kidney graft (KTx group), consisting of patients with a solitary kidney transplantation (n = 13) and patients who received SPK and lost their pancreatic graft due to vascular thrombosis (n = 2) at 2 and 4 days after transplantation. Biochemical markers for endothelial dysfunction including Ang-1, Ang-2 and sTM, together with mucosal capillary density and morphology were compared to 26 DM type 1 patients with nephropathy (DN group) on the waiting list for SPK and DM type 1 patients with an estimated glomerular filtration rate (eGFR) of ≥40 mL/min (n = 15, DM ≥ 40 mL/min group). Patients with active infection, liver failure, active autoimmune disease, epilepsy or malignancy in the last 5 years (except patients treated for basal cell carcinoma that were in full remission) were excluded from the study. In addition, 21 patients from the DN group who underwent SPK were studied longitudinally for 12 months (SPKFU). In this group, SDF and the analysis of endothelial dysfunction markers Ang-1, Ang-2 and sTM were assessed at 1 (M1), 6 (M6) and 12 (M12) months after transplantation. Mean age and duration of diabetes were similar in follow-up patients compared to patients in DN group and SPK group. Twenty healthy and age-matched volunteers served as the control group. None of the control subjects was taking medication.

All transplantations were performed at the Leiden University Medical Center (LUMC), the Netherlands. Organs for the SPK group came from brain-dead donors registered at the national donor registry. The procedure of SPK transplantation has been described previously [23]. All SPK transplants were performed through a midline abdominal incision with both organs placed intraperitoneally. The kidney was placed in the left iliac fossa, with its vessels anastomosed to the common or external iliac vessels in an end-to-side fashion. The ureter urinary bladder anastomosis was performed using the Lich–Gregoir technique. Double-J stenting of the anastomosis was used. The portal vein of the pancreas graft was anastomosed end-to-side to the recipient's vena cava inferior or right iliac vessels. The superior mesenteric and splenic arteries were reconstructed using a donor iliac artery Y graft. In some cases, the pancreas graft was procured with the celiac trunk and superior mesenteric artery on the aorta patch in which no vascular reconstruction was needed. All pancreas grafts were anastomosed end-to-side to the common iliac artery of the recipient. In 22 of the 38 patients (58%) exocrine pancreatic juices were drained via the bladder, followed by enteric conversion and the other patients had a direct enteric drainage. SPK patients used at least two types of immunosuppression. Type of induction immunosuppression during transplantation was dependent on date of transplantation. Patients transplanted before 2008 received induction with interleukin-2 receptor blocker daclizumab (100 mg/day) on the day of transplantation and 10 days after transplantation, followed by triple therapy with prednisone (tapered to a dose of 10 mg by month 3), tacrolimus or cyclosporine and mycophenolatemofetil (MMF; target area under the curve (AUC; 30–60 ng.h/mL). Cyclosporine and tacrolimus AUC were estimated using a population based, two-compartment pharmacokinetic model combined with limited sampling and Bayesian fitting [24-26]. After each AUC assessment, dosage adjustments were made to achieve the predefined target AUC: Cyclosporine AUC: 5400 ng.h/mL within the first 6 weeks, which corresponds with a mean average trough level of 225 ng/mL; after 6 weeks, 3250 ng.h/mL, which corresponds to a mean average trough level of 125 ng/mL; tacrolimus AUC: 210 ng.h/mL within the first 6 weeks, which corresponds to a mean average trough level of 12.5 ng/mL; after 6 weeks, 125 ng.h/mL, which corresponds with a mean average trough level of 7.5 ng/mL. Patients transplanted after 2008 received induction therapy with anti-CD52 antibody alemtuzumab (15 mg/day, subcutaneously) for 2 days followed by glucocorticoid-free therapy with MMF and tacrolimus (n = 12). In the SPKFU group, 20 patients received alemtuzumab as induction therapy and one patient received basiliximab (20 mg/day) at day 0 and 4.

Patients that received a KTx were transplanted in the following way: the iliac vessels were reached through a pararectal incision. The donor's renal vessels were anastomosed end-to-side to the common or external iliac vessels of the recipient. The ureter was anastomosed to the urinary bladder using the Lich–Gregoir technique. Double-J stenting for the vesico-ureteric anastomosis was used [27]. Patients received daclizumab as induction therapy after solitary KTx (100 mg/day on the day of transplantation and 10 days after transplantation) followed by triple therapy with prednisone (tapered to a dose of 10 mg by 6 weeks), tacrolimus (AUC 210 ng.h/mL first 6 weeks, then 125 ng.h/mL) or cyclosporine (AUC 5400 ng.h/mL first 6 weeks then 3250 ng.h/mL) and MMF (AUC 30–60 ng.h/mL). Patients were treated routinely with oral val-ganciclovir prophylaxis for 3 months, except for a CMV negative donor recipient status.

Microcirculatory imaging

The SDF microscan (MicroVision Medical Inc., Wallingford, PA, USA) was performed as described earlier with minor modifications [22]. In short, all patients were measured in supine position in a temperature-controlled room (22°C) by a trained observer. Oral mucosal microvasculature in the labial area was visualized with a 5× objective with a 0.2 NA providing a 325-fold magnification on screen and were sized 720 × 576 pixels (8 bit gray-scale). Ten video files (50 frames each) were obtained of capillary loops in labial mucosa from each quadrant of the lips and saved as Y800-AVI files using IC capture imaging software (The Imaging Source Europe GmbH, Bremen, Germany). The portable, handheld device was connected to a computer and monitor via an analog-to-digital converter (Domino melody framegrabber, Euresyss.a., Angleur, Belgium). A concise review about the SDF imaging technique has been described previously [21].

Analysis of SDF measurements

Before analysis, the video files were anonymized so that the assessor was blinded to the subject's identity. Capillary loops were assessed by two individual assessors in a randomized, blind fashion. From the 40 video files obtained of the microcirculation, the four technically best files were selected from each quadrant of the lip for analysis. Mean vessel density (capillaries/mm2) was calculated by observation of number of vessels per screen shot. Subsequently, tortuosity of capillary loops was assessed according to a validated scoring system described previously and the average of assessed capillary tortuosity was used to calculate mean tortuosity index per patient [22].

Laboratory assessment and endothelial structure evaluation

All persons enrolled in this study underwent routine venous blood sampling before the morning intake of immunosuppression. The following data were evaluated: creatinin, urea, HbA1c, glucose, hemoglobin and proteinuria in 24 h urine. Glomerular filtration rate was calculated with creatinin concentration using the Modification of Diet in Renal Disease (MDRD) formula. Simultaneously, blood was collected for analysis of serum Ang-1, Ang-2 and sTM. Blood collection tubes were centrifuged for 10 min at 3000 rpm after which serum was stored in microcentrifuge tubes at –20°C until required for analysis. Ang-1, Ang-2 and sTM concentrations were measured by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA and Diaclone Research, Besançon, France) according to the manufacturer supplied protocol. The intra- and interassay coefficients of variation were 3.3% and 6.4% for Ang-1, 6.5% and 9.1% for Ang-2 levels and 3.9% and 9.8%, respectively, for sTM levels. Since the Ang-2/Ang-1 ratio, rather than the absolute levels of either cytokine has been considered to determine the functional status of the microvasculature, we calculated this ratio in the different groups [44, 45].

Statistical analyses

Continuous normally distributed data are presented as mean ± standard error of the mean (SEM), unless stated otherwise. Differences between two groups in the cross-sectional study were analyzed using the unpaired two-sample t-test. When criteria for parametric testing were not met, median and interquartile range (IQR) are presented and tested with the Mann–Whitney test. Categorical variables were analyzed by a Chi-square test. In addition, multivariable linear regression was used to adjust for possible confounders. Comparisons of mean differences between the four time points in the longitudinal SPKFU study were performed using repeated measures ANOVA.

Correlations between interval variables were calculated using the Spearman rank correlation. Differences were considered statistically significant with p < 0.05. Data analysis was performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism, version 5.0 (GraphPad Prism Software Inc., San Diego, CA, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. Disclosure
  10. References

Patient characteristics

Baseline subject characteristics of DN, DM ≥ 40 mL/min, SPK and KTx groups in the cross-sectional study are presented in Table 1. There were no significant differences in patient characteristics between the DN and SPK group, with the exception of age, glucose and HbA1c levels, proteinuria and eGFR (p < 0.05). Compared to the DM ≥ 40 mL/min patients, DN group had significantly higher systolic and diastolic blood pressure, decreased eGFR and proteinuria levels and higher HbA1c levels (p < 0.05). As expected, SPK showed improvement in renal function and normalization in blood glucose metabolism compared to DN and DM ≥ 40 mL/min patients (p < 0.05). The median follow-up period of patients after SPK was 45.0 months (IQR 18.5–106.5) and after KTx 38.5 months (IQR 8.5–73.5). Patients in the KTx group had significantly better renal function compared with DN patients and higher glucose and HbA1c levels compared with SPK patients.

Table 1. Patient characteristics of controls, diabetes mellitus Type 1 patients with an eGFR of ≥40 mL/min (DM ≥ 40 mL/min), diabetic nephropathy (DN) patients, simultaneous pancreas–kidney transplantation (SPK) and kidney transplantation (KTx) patients
 ControlsDM ≥ 40 mL/minDNSPKKTx
 (N = 20)(N = 15)(N = 26)(N = 38)(N = 15)
  1. All data are mean ± SD, unless otherwise specified.

  2. a

    The patients in this group who received SPK lost their pancreas graft after 2 and 4 days following transplantation (n = 2).

  3. 1p < 0.05 versus DN group; 2p < 0.05 versus DN and SPK group; 3p < 0.05 versus DM ≥ 40 mL/min; 4p < 0.05 versus SPK; 5p < 0.05 versus KTX.

  4. ACE = angiotensin converting enzyme; BMI = body mass index; BP = blood pressure; eGFR = estimated glomerular filtration rate; IQR = interquartile range; SPK = simultaneous pancreas–kidney transplantation.

Age (years)44.8±11354.6±1343.8±6348.7±91, 347.6±10
Sex, male, N (%)10(50%)6(40%)18(72%)25(66%)6(40%)
Smoking, N (%)0(0%)2(15%)0(0%)3(8%)1(7%)
Median time since transplantation (months) (IQR)   45.0(18.5–106.5)38.5(8.5–73.5)a
Duration diabetes (years) 35.5±1029.3±827.9±934.9±9
BMI (kg/m2)25.3±423.8±325.2±324.4±425.0±5
Dialysis, N (%) 0(0%)4(5%)0(0%)0(0%)
Systolic BP (mmHg)135±18130±13145±203141±25138±29
Diastolic BP (mmHg)83±9371±886±11382±13381±143
eGFR (mL/min/1,73 m2)92.7±162, 3, 569.9±2423.9±20352.7±191, 362.0±231
Median proteinuria (g/24 h) (IQR) 0.3(0.1–0.3)0.7(0.3–1.5)30.3(0.2–0.8)10.2(0.2–0.4)2
HbA1c (%) 7.1±18.8± 235.6±1 1, 38.5± 12,3
Glucose (mmol/L)5.3±11, 3, 512.8±513.3±65.8±3 1, 313±74
Hemoglobin (mmol/L)8.7±128.2±17.6±18.1±18.5±1
Rejection after SPK or KTx, N (%)   14(37%)1(7%)
Diabetes after SPK, N (%)   3(8%)  
Acetylsalicylic acid, N (%) 4(27%)4(15%)11(29%)3(20%)
Antihypertensives, N (%)         
 ACE inhibitor  0(0%)16(64%)15(40%)7(47%)
 Diuretics  5(33%)13(52%)9(24%)14(27%)
 β-blockers  0(0%)10(40%)319(50%)36(40%)3
 Calcium antagonists  2(13%)11(44%)323(61%)37(47%)3
 Angiotensin-II antagonists  3(20%)13(52%)38(21%)10(0%)1
Statines, N (%) 8(53%)16(60%)27(71%)5(33%) 4
Steroid-free, Alemtuzumab induction, N (%)   12(13%) 
Immunosuppressive, N (%)       
 Cyclosporine      13(34%)1(7%)4
 Tacrolimus      25(66%)12(80%)
 Prednisone      27(71%)9(60%)
 Azathioprine      3(8%)0(0%)
 Everolimus      2(5%)0(0%)
 Sirolimus      0(0%)1(7%)
 Mycophenolate mofetil      28(74%)14(93%)

More than 2 years after transplantation, three patients developed diabetes type II in the SPK group of which two were treated with oral antidiabetic medication and one patient with insulin. Fourteen of the 38 (37%) patients experienced acute rejection after SPK, which is similar to rejection rates reported in other studies after SPK [28]. Of these, 10 patients had a renal biopsy proven interstitial acute rejection, one of these patients had also vascular rejection. Four patients were treated as rejection, without prior transplant biopsy. Seven patients were successfully treated with solumedrol, six patients with solumedrol and antithymocyte globulin (ATG) and one patient with ATG alone. In the KTx group, no patients developed rejection after transplantation.

The clinical characteristics of the patients in the longitudinal SPKFU group are shown in Table 2. As expected, patients showed normalization of glucose levels (p = 0.04) and eGFR (p = 0.006) at 1 month after SPK. Since all the patients in the SPKFU group were transplanted after 2008, immunosuppressive therapy was initially glucocorticosteroid-free, with MMF and tacrolimus as maintenance therapy. Twelve months after SPK, two patients were converted to everolimus due to side effects of tacrolimus. In four patients treatment with prednisone was added at the time of conversion from tacrolimus to cyclosporine due to side effects caused by tacrolimus (two patients) or MMF (two patients). In this group, five patients experienced interstitial rejection and one patient had also vascular rejection after transplantation. These patients were treated with prednisolone and ATG. Six months after SPK, two patients developed diabetes type II, which was treated with oral antidiabetic medication.

Table 2. Patient characteristics in SPKFU patients at 1 (M1), 6 (M6) and 12 (M12) months after SPK transplantation
 M1aM6M12
Characteristic(N = 21)(N = 21)(N = 21)
  1. All data are mean ± SD, unless otherwise specified.

  2. *p < 0.05 versus DN from Table 1.

  3. a

    Patients were recruited from the DN group (see Table 1 for patient characteristics).

  4. ACE = angiotensin converting enzyme; BMI = body mass index; BP = blood pressure; eGFR = estimated glomerular filtration rate; IQR = interquartile range; SPK = simultaneous pancreas–kidney transplantation.

Age (years)44.0± 644.4± 645.0± 6
Sex, male N (%)16(76%)  
Smoking, N (%)0 0 0 
BMI (kg/m2)23.8*± 324.0± 224.1± 3*
Dialysis, N (%)0 0 0 
Systolic BP (mmHg)123± 22132± 23130± 16
Diastolic BP (mmHg)75± 13777± 1378± 6
eGFR (mL/min/1,73 m2)56.5± 23 *55.2± 15 *54.1± 10 *
Median proteinuria (g/24 h) (IQR)0.3(0.3–1.2)0.3(0.2–1.0)0.3(0.1–0.7)
HbA1c (%)6.7± 25.3± 0 *5.4± 0 *
Glucose (mmol/L)6.2± 1*5.3± 1*5.7± 1*
Hemoglobin (mmol/L)6.6± 17.4± 17.9± 1
Rejection after transplantation, N (%)0 3(14%)2(10%)
Diabetes after SPK, N (%)1(5%)2(10%)0 
Acetylsalicylic acid, N (%)3(14%)1(5%)4(19%)
Antihypertensives, N (%)      
 ACE inhibitor2(10%)3(14%)4(19%)
 Diuretics0(0%)1(5%)2(10%)
 β-blockers7(33%)4(19%)4(19%)
 Calcium antagonists4(19%)5(24%)8(38%)
 Angiotensin-II antagonists0 0 0 
Statines, N (%)2(10%)2(10%)3(14%)
Steroid-free, alemtuzumab induction, N (%)20(95%)  
Immunosuppressive, N (%)      
 Tacrolimus18(86%)17(81%)16(76%)
 Ciclosporine2(5%)3(14%)3(14%)
 Prednisone1(5%)6(29%)6(29%)
 Mycophenolate mofetil21(100%)20(95%)20(95%)
 Everolimus1(5%)1(5%)2(10%)

Reversibility in capillary tortuosity after SPK

There was no difference in the capillary density in the cross-sectional study between the DN (mean 19.88 ± 4.1, SEM), DM ≥ 40 mL/min (mean 19.03 ± 0.5, SEM), SPK (mean 20.79 ± 3.1, SEM), control (mean 21.34 ± 3.1, SEM) and KTx (mean 19.14 ± 0.5, SEM) group. However, the morphology of the capillaries in the DN and DM ≥ 40 mL/min patients was significantly disturbed compared to the controls (Figure 1A). We observed significantly more capillary tortuosity in the DN patients (mean 1.83 ± 0.4, SEM, p < 0.001) and DM ≥ 40 mL/min patients (mean 1.55 ± 0.1, SEM, p < 0.0001) compared to the controls (mean 1.15 ± 0.2, SEM). After KTx (mean 1.64 ± 0.1, SEM) there was no significant decrease in capillary tortuosity compared to DN (p = 0.06) and DM ≥ 40 mL/min (p = 0.3656) group. However, interestingly, SPK (mean 1.31 ± 0.3, SEM, p < 0.001) showed reversal in capillary tortuosity compared to DN and DM ≥ 40 mL/min group (Figure 1B). The three patients who developed diabetes type II after SPK did not show more increased capillary tortuosity compared to the other patients in the SPK group.

image

Figure 1. (A) Sidestreamdarkfield images of the oral mucosa visualizing the microvascular capillaries of a representative patient in the control, DM ≥ 40 mL/min, DN, SPK and KTx group. Black arrows: capillary loops. (B) Mean tortuosity index of microvascular capillaries in the control (n = 20), DM ≥ 40 mL/min (n = 15), DN (n = 26), SPK (n = 38) and KTx (n = 15) group. Data shown are mean ± SEM. *p < 0.05.

Download figure to PowerPoint

Next, we investigated capillary density and morphology longitudinally in the SPKFU group before, 1, 6 and 12 months after SPK. No difference was observed in capillary density before and after SPK. Most importantly, SPK showed reversal of capillary tortuosity within 1 year after SPK, reaching significance at 6 and 12 months after transplantation (mean 1.52 ± 0.1, SEM and 1.23 ± 0.0 vs. DN 1.83 ± 0.4, p < 0.01 and p < 0.001, respectively, Figures 2A and B). The differences remained significant in both the cross-sectional and SPKFU group after adjustment for age, sex, body mass index (BMI), systolic and diastolic blood pressure and smoking.

image

Figure 2. (A) Sidestream dark field images of the oral mucosa visualizing the microvascular capillaries of a representative DN patient before (DN), 1 (M1), 6 (M6) and 12 (M12) months after SPK in the longitudinal SPKFU group. Black arrows: capillary loops. (B) Longitudinal course of the mean tortuosity index of microvascular capillaries in DN patients before (DN, n = 26), 1 (M1, n = 21), 6 (M6, n = 21) and 12 (M12, n = 21) months after SPK in the SPKFU group. Data shown are mean ± SEM. *p < 0.01, M6 compared to DN; **p < 0.001, M12 compared to DN.

Download figure to PowerPoint

Correlation between tortuosity, Hb1Ac, renal function and previous rejection

Next, correlation analyses in the SPK patients was performed for the capillary density and tortuosity index with several factors that are known to influence the microvasculature, including time since transplantation, eGFR, HbA1c, proteinuria and previous rejection. No correlation was found between capillary density and time since transplantation, eGFR, HbA1c, proteinuria and previous rejection. There was a significant correlation between tortuosity with eGFR (r = –0.26, p = 0.005), Hb1Ac levels (r = 0.40, p < 0.0001) and previous rejection (p = 0.0160). There was no association between tortuosity index and time since transplantation or proteinuria (data not shown).

Reversal to baseline of Ang-2/Ang-1 ratio after SPK

To evaluate the effects of SPK on the microvascular conditions, we also measured serum levels of sTM, Ang-1 and Ang-2 and calculated the Ang-2/Ang-1 ratio as markers for endothelial dysfunction. We detected a significant decrease in sTM levels in patients after SPK (mean 9.7 ± 1 ng/mL, SEM, p = 0.004) and KTx (mean 6.3 ± 1 ng/mL, SEM, p < 0.0001) compared to DN patients (mean 15.2 ± 1 ng/mL, SEM, p = 0.004; Figure 3A). In the SPKFU group, sTM levels started to decrease at 1 month (mean 13.35 ± 2.0 ng/mL, SEM) after transplantation and remained low after 6 months (mean 11.83 ± 1.6 ng/mL, SEM), reaching significance at 12 months (10.42 ± 1.2 ng/mL, SEM) as compared to before SPK (mean 15.2 ± 1 ng/mL, SEM, p < 0.01; Figure 3B). In addition, our results demonstrate normalization in Ang-2/Ang-1 ratio after SPK (mean 0.08 ± 0.02, SEM) compared to the DN (mean 0.16 ± 0.04, SEM, p = 0.04) and DM ≥ 40 mL/min patients (mean 0.16 ± 0.04, SEM, p = 0.03), but no significant decrease after KTx (mean 0.20 ± 0.04, SEM, p = 0.6219 and p = 0.5437, respectively; Figure 3C). In the longitudinal study, the Ang-2/Ang-1 ratio started to show a decrease at 6 months (mean 0.09 ± 0.03, SEM) after SPK and remained decreased at 12 months (mean 0.09 ± 0.02, SEM) following transplantation compared to before SPK (mean 0.16 ± 0.04, SEM), however, statistical significance was not reached (p = 0.13 and p = 0.14, respectively; data not shown).

image

Figure 3. (A) Soluble thrombomodulin serum levels in the control (n = 20), DM ≥ 40 mL/min (n = 15), DN (n = 26), SPK (n = 38) and KTx (n = 15) group; *p < 0.05. (B) Soluble thrombomodulin serum levels in DN patients before (DN), 1 (M1), 6 (M6) and 12 (M12) months after SPK in the SPKFU group; *p < 0.05 compared to DN. (C) Ang-2/Ang-1 ratio in the control (n = 20), DM ≥ 40 mL/min (n = 15), DN (n = 26), SPK (n = 38) and KTx (n = 15) group. Data shown are mean ± SEM. *p < 0.05.

Download figure to PowerPoint

Correlation of endothelial function markers with capillary tortuosity, renal function and HbA1c

Next, the correlation between the different endothelial dysfunction markers, capillary density and morphology, eGFR, HbA1c, previous rejection and time since transplantation was analyzed. No correlation between endothelial dysfunction markers and capillary density or tortuosity was found. Moreover, there was no correlation between time since transplantation, previous rejection and endothelial dysfunction markers. Increased Ang-2 and sTM levels correlated significantly with decreased eGFR (r = –0.53, p < 0.0001 and r = –0.28, p = 0.0083) and high Hb1Ac levels with increased Ang-2 (r = 0.23, p = 0.0319). Interestingly, Ang-2/Ang-1 ratio correlated significantly with capillary tortuosity (r = 0.21, p = 0.0348), HbA1c (r = 0.32, p = 0.0038), eGFR (r = –0.29, p = 0.0460) and proteinuria levels (r = 0.2640, p = 0.0365; data not shown) .

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. Disclosure
  10. References

This study shows increased microvascular tortuosity using SDF imaging, and a dysbalance in Ang-2/Ang-1 ratio in DM type 1 patients. Interestingly, we demonstrated reversal of capillary tortuosity and normalization of the Ang-2/Ang-1 ratio after SPK, but not after KTx alone. Furthermore, increased microvascular tortuosity correlated with increased Ang-2/Ang-1 ratio. Importantly, the longitudinal study demonstrated that both reversibility of microvascular damage and decrease in sTM and Ang-2/Ang-1 balance occurred in the first year after SPK. These findings suggest that SPK is effective in reversing systemic microvascular complications in DN patients early after transplantation. Assessment of capillary tortuosity, as marker for microvascular disease, using the noninvasive SDF imaging can be used to estimate the degree of microvascular derangements in DN patients before and after SPK.

Microvascular disease is one of the most important drivers of diabetic complications [29, 30]. Several mechanisms have been described for the pathogenesis of microvascular disorders in patients with DM. The capillaries in diabetic patients tend to be leaky and tortuous, lacking the hierarchical arrangement of arteries, capillaries and venules due to perivascular stromal cell loss [31-33]. Hyperglycemia in particular can initiate disturbances in blood flow and afflict the interaction between perivascular stromal cells and microvascular endothelial cells. Hereby, the loss of perivascular stromal cells leads to loss of its primary functions, including autoregulation of the vessel integrity and compensatory mechanisms for the fluctuating hydrostatic pressure. Specifically, this impaired autoregulation of the microvasculature could result in disruption of the basement membrane and the failure to maintain the stability of the vessel wall against irregular longitudinal traction and transmural pressure leading to dilated and tortuous vessels [32-38]. Ang-2 has been assumed to destabilize vessels by promoting the weakening of perivascular stromal–endothelial cell interaction [39, 40].

The increased microvascular tortuosity in diabetes as observed in the present study concurs with previous data on microvascular alterations in diabetic patients as determined by skin capillaroscopy [41]. Similarly, previous clinical observations using conventional capillaroscopy revealed that diabetic retinopathy appeared to be associated with increased tortuosity as well, suggesting that this might be an early sign of microvascular damage in diabetes [33]. However, compared to conventional capillaroscopy, SDF imaging has the advantage to assess the microvasculature without injecting fluorescent dyes and it enables measurement of the superficial skin and mucous microcirculation in a noninvasive manner [21]. We previously could demonstrate, using SDF measurements, that the presence of microvascular tortuosity is associated with macrovascular disease in diabetics [22]. However, the extensiveness of microvascular abnormalities has not been measured in DN patients before and after SPK using SDF imaging and correlation with markers for endothelial dysfunction and clinical features has not been performed yet.

Previous clinical studies showed that 10 years of normoglycemia after pancreas transplantation ameliorated the glomerular and tubular lesions that characterize DN in patients with long-term type 1 diabetes who did not receive renal grafts [42]. Additional studies provided evidence for improvement of diabetic polyneuropathy after pancreatic transplantation as well [43, 44]. Although we observed reversal of microvascular tortuosity after SPK, this study did not explore the mechanisms behind these improvements.

Increased microvascular tortuosity in DN coincided with increased levels of sTM and a disturbed Ang-2/Ang-1 balance, which is in line with previous studies that have demonstrated a positive correlation with microvascular destabilization and these markers of endothelial dysfunction [45, 46, 49]. Ang-2 is a competitive ligand for the same Tie-2 receptor as for Ang-1, with competing, antagonistic effects on angiogenesis and microvascular remodeling. Increased Ang-2/Ang-1 ratio has been shown to be associated with hyperglycemia, chronic kidney disease, acute coronary syndrome, sepsis and variety of diseases known for their common characteristic of endothelial dysfunction and microvascular inflammation (45-48). The Ang-2/Ang-1 ratio showed only normalization after SPK, while it was still increased in the presence of diabetes and also was not affected by kidney transplantation alone. In addition, the Ang-2/Ang-1 ratio correlated to capillary tortuosity underscoring the systemic nature of microvascular destabilization in diabetes.

The strength of our study was the use of a noninvasive tool to visualize the microvasculature in a study of SPK and DN patients before and after transplantation. However, our study has also some limitations. In this studied cohort there were no patients who received a solitary kidney transplant that could be followed longitudinally. Therefore, future longitudinal studies should possibly also include such patients to further corroborate the potential contribution of reversal of diabetes on the microcirculation.

In conclusion, the present study revealed a disturbed microvascular morphology in DM type 1 patients and SPK resulted in reversal of systemic microvascular derangements and normalization of markers for endothelial dysfunction. The use of SDF imaging allows for easy noninvasive and sequential monitoring of microvascular disease in patients with diabetes.

Author contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. Disclosure
  10. References

M.K. participated in the design and coordination of the study and wrote the manuscript. B.M.B. contributed to the discussion. E.L., together with M.K. carried out the experiments. H.C.dB. contributed to discussion. M.M. contributed to the statistical analysis. A.F.S., P.J.M. vdB. and J.I.R. contributed to the discussion. E.J.P.dK. and J.W.dF. participated in the design and discussion. A.J.vZ. contributed to the discussion. T.J.R. and M.E.J.R. contributed to the design and supervision of the study and helped to draft the manuscript. All authors read and approved the final manuscript.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. Disclosure
  10. References

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Author contributions
  9. Disclosure
  10. References
  • 1
    Cumbie BC, Hermayer KL. Current concepts in targeted therapies for the pathophysiology of diabetic microvascular complications. Vasc Health Risk Manag 2007; 3: 823832.
  • 2
    Tooke JE. Microvasculature in diabetes. Cardiovasc Res 1996; 32: 764771.
  • 3
    Rabelink TJ, de Boer HC, van Zonneveld AJ. Endothelial activation and circulating markers of endothelial activation in kidney disease. Nat Rev Nephrol 2010; 6: 404414.
  • 4
    Iwashima Y, Sato T, Watanabe K, et al. Elevation of plasma thrombomodulin level in diabetic patients with early diabetic nephropathy. Diabetes 1990; 39: 983988.
  • 5
    Oida K, Takai H, Maeda H, et al. Plasma thrombomodulin concentration in diabetes mellitus. Diabetes Res Clin Pract 1990; 10: 193196.
  • 6
    Fiedler U, Augustin HG. Angiopoietins: A link between angiogenesis and inflammation. Trends Immunol 2006; 27: 552558.
  • 7
    Hammes HP, Lin J, Renner O, et al. Pericytes and the pathogenesis of diabetic retinopathy. Diabetes 2002; 51: 31073112.
  • 8
    Jukema JW, Smets YF, van der Pijl JW, et al. Impact of simultaneous pancreas and kidney transplantation on progression of coronary atherosclerosis in patients with end-stage renal failure due to type 1 diabetes. Diabetes Care 2002; 25: 906911.
  • 9
    Smets YF, Westendorp RG, van der Pijl JW, et al. Effect of simultaneous pancreas-kidney transplantation on mortality of patients with type-1 diabetes mellitus and end-stage renal failure. Lancet 1999; 353: 19151919.
  • 10
    Sollinger HW, Odorico JS, Becker YT, D'Alessandro AM, Pirsch JD. One thousand simultaneous pancreas-kidney transplants at a single center with 22-year follow-up. Ann Surg 2009; 250: 618630.
  • 11
    Becker BN, Brazy PC, Becker YT, et al. Simultaneous pancreas-kidney transplantation reduces excess mortality in type 1 diabetic patients with end-stage renal disease. Kidney Int 2000; 57: 21292135.
  • 12
    Bohman SO, Tyden G, Wilczek H, et al. Prevention of kidney graft diabetic nephropathy by pancreas transplantation in man. Diabetes 1985; 34: 306308.
  • 13
    Nyumura I, Honda K, Babazono T, et al. A long-term prevention of diabetic nephropathy in a patient with type 1 diabetes after simultaneous pancreas and kidney transplantation. Clin Transplant 2009; 23(Suppl 20): 5457.
  • 14
    Tyden G, Bolinder J, Solders G, Brattstrom C, Tibell A, Groth CG. Improved survival in patients with insulin-dependent diabetes mellitus and end-stage diabetic nephropathy 10 years after combined pancreas and kidney transplantation. Transplantation 1999; 67: 645648.
  • 15
    Wilczek HE, Jaremko G, Tyden G, Groth CG. Evolution of diabetic nephropathy in kidney grafts. Evidence that a simultaneously transplanted pancreas exerts a protective effect. Transplantation 1995; 59: 5157.
  • 16
    Brekke IB, Ganes T, Syrdalen P, Egge K, Dyrbekk D, Flatmark A. Combined renal and pancreatic transplantation: Effects on advanced diabetic neuropathy and retinopathy. Life Support Syst 1985; 3(Suppl 1): 680684.
  • 17
    Mehra S, Tavakoli M, Kallinikos PA, et al. Corneal confocal microscopy detects early nerve regeneration after pancreas transplantation in patients with type 1 diabetes. Diabetes Care 2007; 30: 26082612.
  • 18
    Navarro X, Sutherland DE, Kennedy WR. Long-term effects of pancreatic transplantation on diabetic neuropathy. Ann Neurol 1997; 42: 727736.
  • 19
    Pearce IA, Ilango B, Sells RA, Wong D. Stabilisation of diabetic retinopathy following simultaneous pancreas and kidney transplant. Br J Ophthalmol 2000; 84: 736740.
  • 20
    Solders G, Wilczek H, Gunnarsson R, Tyden G, Persson A, Groth CG. Effects of combined pancreatic and renal transplantation on diabetic neuropathy: A two-year follow-up study. Lancet 1987; 2: 12321235.
  • 21
    Goedhart PT, Khalilzada M, Bezemer R, Merza J, Ince C. Sidestream Dark Field (SDF) imaging: A novel stroboscopic LED ring-based imaging modality for clinical assessment of the microcirculation. Opt Express 2007; 15: 1510115114.
  • 22
    Djaberi R, Schuijf JD, de Koning EJ, et al. Non-invasive assessment of microcirculation by sidestream dark field imaging as a marker of coronary artery disease in diabetes. Diab Vasc Dis Res 2012. Epub ahead of print.
  • 23
    Marang-van de Mheen PJ, Nijhof HW, Khairoun M, Haasnoot A, van der Boog PJ, Baranski AG. Pancreas-kidney transplantations with primary bladder drainage followed by enteric conversion: Graft survival and outcomes. Transplantation 2008; 85: 517523.
  • 24
    Cremers SC, Scholten EM, Schoemaker RC, et al. A compartmental pharmacokinetic model of cyclosporin and its predictive performance after Bayesian estimation in kidney and simultaneous pancreas-kidney transplant recipients. Nephrol Dial Transplant 2003; 18: 12011208.
  • 25
    Scholten EM, Cremers SC, Schoemaker RC, et al. AUC-guided dosing of tacrolimus prevents progressive systemic overexposure in renal transplant recipients. Kidney Int 2005; 67: 24402447.
  • 26
    Rowshani AT, Scholten EM, Bemelman F, et al. No difference in degree of interstitial Sirius red-stained area in serial biopsies from area under concentration-over-time curves-guided cyclosporine versus tacrolimus-treated renal transplant recipients at one year. J Am Soc Nephrol 2006; 17: 305312.
  • 27
    Khairoun M, Baranski AG, van der Boog PJ, Haasnoot A, Mallat MJ, Marang-van de Mheen PJ. Urological complications and their impact on survival after kidney transplantation from deceased cardiac death donors. Transpl Int 2009; 22: 192197.
  • 28
    Malaise J, Arbogast H, Illner WD, et al. Simultaneous pancreas-kidney transplantation: Analysis of rejection. Transplant Proc 2005; 37: 28562858.
  • 29
    Cheung AT, Perez RV, Chen PC. Improvements in diabetic microangiopathy after successful simultaneous pancreas-kidney transplantation: A computer-assisted intravital microscopy study on the conjunctival microcirculation. Transplantation 1999; 68: 927932.
  • 30
    Kuryliszyn-Moskal A, Dubicki A, Zarzycki W, Zonnenberg A, Gorska M. Microvascular abnormalities in capillaroscopy correlate with higher serum IL-18 and sE-selectin levels in patients with type 1 diabetes complicated by microangiopathy. Folia Histochem Cytobiol 2011; 49: 104110.
  • 31
    Bergers G, Song S. The role of pericytes in blood-vessel formation and maintenance. Neuro Oncol 2005; 7: 452464.
  • 32
    Boone MI, Farber ME, Jovanovic-Peterson L, Peterson CM. Increased retinal vascular tortuosity in gestational diabetes mellitus. Ophthalmology 1989; 96: 251254.
  • 33
    Sasongko MB, Wong TY, Nguyen TT, Cheung CY, Shaw JE, Wang JJ. Retinal vascular tortuosity in persons with diabetes and diabetic retinopathy. Diabetologia 2011; 54: 24092416.
  • 34
    Dobrin PB, Schwarcz TH, Baker WH. Mechanisms of arterial and aneurysmal tortuosity. Surgery 1988; 104: 568571.
  • 35
    Hanahan D. Signaling vascular morphogenesis and maintenance. Science 1997; 277: 4850.
  • 36
    Jackson ZS, Dajnowiec D, Gotlieb AI, Langille BL. Partial off-loading of longitudinal tension induces arterial tortuosity. Arterioscler Thromb Vasc Biol 2005; 25: 957962.
  • 37
    Kohner EM, Patel V, Rassam SM. Role of blood flow and impaired autoregulation in the pathogenesis of diabetic retinopathy. Diabetes 1995; 44: 603607.
  • 38
    Kristinsson JK, Gottfredsdottir MS, Stefansson E. Retinal vessel dilatation and elongation precedes diabetic macular oedema. Br J Ophthalmol 1997; 81: 274248.
  • 39
    Cai J, Kehoe O, Smith GM, Hykin P, Boulton ME. The angiopoietin/Tie-2 system regulates pericyte survival and recruitment in diabetic retinopathy. Invest Ophthalmol Vis Sci 2008; 49: 21632171.
  • 40
    Hughes S, Gardiner T, Baxter L, Chan-Ling T. Changes in pericytes and smooth muscle cells in the kitten model of retinopathy of prematurity: Implications for plus disease. Invest Ophthalmol Vis Sci 2007; 48: 13681379.
  • 41
    Meyer MF, Pfohl M, Schatz H. Assessment of diabetic alterations of microcirculation by means of capillaroscopy and laser-Doppler anemometry. Med Klin (Munich) 2001; 96: 7177.
  • 42
    Fioretto P, Steffes MW, Sutherland DE, Goetz FC, Mauer M. Reversal of lesions of diabetic nephropathy after pancreas transplantation. N Engl J Med 1998; 339: 6975.
  • 43
    Kennedy WR, Navarro X, Goetz FC, Sutherland DE, Najarian JS. Effects of pancreatic transplantation on diabetic neuropathy. N Engl J Med 1990; 322: 10311037.
  • 44
    Martinenghi S, Comi G, Galardi G, Di CV, Pozza G, Secchi A. Amelioration of nerve conduction velocity following simultaneous kidney/pancreas transplantation is due to the glycaemic control provided by the pancreas. Diabetologia 1997; 40: 11101112.
  • 45
    David S, John SG, Jefferies HJ, et al. Angiopoietin-2 levels predict mortality in CKD patients. Nephrol Dial Transplant 2012; 27: 18671872.
  • 46
    Tuo QH, Zeng H, Stinnett A, et al. Critical role of angiopoietins/Tie-2 in hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis. Am J Physiol Heart Circ Physiol 2008; 294: H2547H2557.
  • 47
    David S, Kumpers P, Hellpap J, et al. Angiopoietin 2 and cardiovascular disease in dialysis and kidney transplantation. Am J Kidney Dis 2009; 53: 770778.
  • 48
    David S, Kumpers P, Lukasz A, et al. Circulating angiopoietin-2 levels increase with progress of chronic kidney disease. Nephrol Dial Transplant 2010; 25: 25712576.
  • 49
    Keven K, Elmaci S, Sengul S, et al. Soluble endothelial cell protein C receptor and thrombomodulin levels after renal transplantation. Int Urol Nephrol 2010; 42: 10931098.