Hemorheology in kidney transplantation: A role for cardiovascular risk?

1 Introduction

Proper tissue perfusion depends on correct blood flow and hemorheological properties of blood elements; their alterations, through endothelial damage, play a significant role in subsequent fibrosis with progression to end-stage injury [18]. Uremic syndrome, namely end stage renal disease, is a systemic condition with related hemorheological changes, such as a plasma viscosity (ηP) increase and erythrocyte deformability (ED) decrease with a still normal whole blood viscosity due to decreased haematocrit-value (Ht). Chronic inflammation, lipid oxidation, endothelial dysfunction, hyperparathyroidism, uremic toxins, aluminum overload, acidosis, membrane-ATP-ase dysfunction and alterations in nitric oxide metabolism have been indicated as causative factors for these hemorheological changes [4, 10]. Most uremia-related factors are poorly corrected by hemodialysis and medical supporting therapy resulting in a persistent cardiovascular risk 20-fold higher in comparison to general population, and in cardiovascular events still being the leading cause of death in these patients [5]. As for hemorheological changes, hemodialysis fails to correct alterations and, on the contrary, worsens some of them [14]. Kidney transplantation reduces the overall 4-year risk of death and cardiovascular events respectively to 68% and 48–82%, but cardiovascular risk remains still higher than in general population, and it is the first cause of death among kidney transplanted patients (KT) [3, 13]. Once again, hemorheological changes could enter the group of unconventional risk factors playing a role on this increased risk [3]. Literature provides few and heterogeneous data about hemorheology in KT: Koppensteiner et al. pointed out an abnormal red cell aggregation [11], Linde et al. found a reduced ED in KT [12], Zanazzi et al. found alteration of all hemorheological parameters [20], Hammes et al. described an impairment in the microvascular blood flow of KT patients related to an abnormal regulation of asymmetric dimethylarginine release [8]. Focusing on immunosuppressive drugs, a possible detrimental role has been assumed for Cyclosporin (CyA) on ED [2, 9] and Akoglu et al. has reported a more significant decrease of ED in KT from CyA use in comparison to Tacrolimus (FK) [1].

2 Methods

Our aim is to characterize the hemorheology of KT in comparison to uremic patients and healthy control volunteers (HV). All participants were asked to sign informed consent before obtaining blood specimens for routine and hemorheology measures. This research complies with the requirement for ethical publication in Clincal Hemorheology and Microcirculation as published in Clin HemorheolMicrocirc. 2010;44(1):1-2.

3 Selection and description of participants

We considered three groups: HV; uremic patients undergoing intermittent hemodialysis (HD), where blood samples were collected before (pre-HD) and after (post-HD) the second dialysis session of the week; KT. Demographic characteristics are summarized in Table 1. KT were receiving immunosuppressive treatment as follows: 75 patients were receiving calcineurin inhibitors (cnI) and 11 patients were receiving a combination of cnI and mTor-I (44 on CyA and 42 on FK); 22 patients were receiving mTor inhibitors (mTor-i).

4 Technical information

All measurements were performed with a coaxial cylinder rehometer (Haake RV20-CV100, Haake, Germany) at even temperature 37°C, according with International Committee for Standardization in Haematology [19]. Plasma viscosity (ηP) was measured at 300 Hz-shear rate. Whole blood viscosity was measured at 1 Hz-shear rate (ηS₁) and at 200 Hz-shear rate (ηS₂₀₀) and the values were standardized to Ht 40%. Erythrocyte aggregation index (EAI) was evaluated as ηS₁/ηS₂₀₀ ratio. Yield stress (τ₀) was measured through the Casson regression model [15]. Erythrocyte deformability (ED) was evaluated as Taylor factor [18] (Tk) [1-(ηP/ηS₂₀₀)0.4/Ht], index of erythrocyte stiffness. We evaluated viscous-elastic behavior of blood in terms of shear elastic modulus (G’), defined as the ratio of stress to strain (G’ =σ/γ), applying a 8% strain under oscillatory flow regimen.

5 Statistical techniques

Statistical analysis was performed with GraphPad Prism 6 software. Outlier values were eliminated. Distribution of data was assessed with D’Agostino & Pearson omnibus normality test; data were expressed as median (25% percentile –75% percentile); differences were assessed with non-parametric Mann-Whitney test with a two-tailed p value <0,05 (95% confidence level). Difference between G’ slopes was assessed with linear regression (95% confidence level for slopes).

6 Results

Results are summarized in Fig. 1. In the following exposition results are expressed as medians (25% percentile –75% percentile). ηP was increased in HD, especially post-HD (pre-HD vs post-HD: 1,59 (1,37-1,72) vs 1,80 (1,55-1,96) mPa*sec; p < 0,0001); it resulted lower in KT (KT vs pre-HD: 1,33 (1,26-1,41) vs 1,59 (1,37-1,72) mPa*sec; p < 0,0001). Plasma levels of albumin and other protein fractions in HD and KT groups are expressed in Table 1. ηS ₁ resulted considerably lower in KT when compared to HD (KT vs pre-HD: 12,48 (9,76-14,37) vs 23,14 (16,89-34,97) mPa*sec; p < 0,0001), but it was higher than in HV (HV vs KT: 9,68 (7,83-14,82) vs 12,48 (9,76-14,37) mPa*sec; p < 0,05). EAI resulted lower in KT (KT vs pre-HD: 3,20 (2,66-3,77) vs 5,60 (4,35-7,71); p < 0,0001). τ ₀ was lower in KT (KT vs pre- HD: 0,10 (0,09-0,12) vs 0,17 (0,13-0,23) mPa; p < 0,0001). ηS ₂₀₀ was lower in KT (KT vs pre-HD: 3,94 (3,6-4,22) vs 4,71 (3,83-5,43) mPa*sec; p < 0,0001), but it was higher than in HV (HV vs KT: 3,63 (3,06-3,99) vs 3,94 (3,6-4,22) mPa*sec; p 0,0005). Tk was markedly higher in KT than in HD (KT vs pre-HD: 0,86 (0,83-0,90) vs 0,81 (0,78-0,86); p < 0,0001) and HV (HV vs KT: 0,77 (0,70-0,83) vs 0,86 (0,83-0,90); p < 0,0001). Analysis of viscous-elastic blood behaviour confirmed higher G’ in KT than in HD and HV (Linear Regression, 95% confidence interval for slopes: Controls 0,007445 to 0,01497, pre-HD 0,01595 to 0,02663, KT 0,05813 to 0,06353); see Fig. 2. As for the ID, we have not found differences of all hemorheological parameters in KT treated with cnI and KT cnI-free. Among cnI-treated KT, we have not found differences from patients receiving CyA with those receiving FK.

7 Discussion

Our results confirm an alteration of hemorheological parameters in pre-HD. Moreover, ηP, ηS₁, ηS₂₀₀, EAI and τ₀ further worsen post-HD, because of both dehydration and acute phase proteins rising. KT corrects many of these alterations. ηP was normal in KT, unless albumin and α1-globulins levels were higher than in HD patients. It is likely that the lower levels of γ-globulins allowed the decrease of ηP in KT, whilst reciprocal interactions between protein fractions may have increased ηP in HD. ηS ₁ and ηS ₂₀₀ simulate blood behavior in large and small vessels respectively. ηS ₁ is highly influenced by erythrocyte aggregation and plasma viscosity, and resulted markedly lower in KT, as was for EAI. Theoretically, this allows physiologic formation of erythrocyte rouleaux in low blood flow areas (such as venules) and the maintenance of a physiologic laminar flow; it represents a protective mechanism, reducing thrombotic risk, rise in peripheral resistance, hypoxia and tissue damage [7]. ηS ₂₀₀ was reduced in KT, but levels remained higher than in HV. This could be ascribed to a low ED, since at high velocity rate erythrocytes are completely disaggregated and blood viscosity is mainly determined by ED. τ ₀ was lower in KT. For any liquid suspension, τ₀ represents the minimum stress to be applied in order to obtain flow; as for the blood, τ₀ represents the minimum strength required to break interactions between erythrocytes. Afterwards, τ₀ is inversely related to EAI. During the isometric-systole, an increase of τ₀ causes a rise in the cardiac after-load; therefore, it chronically contributes to myocardial hypertrophy. Tk, inversely related to ED, evaluates the erythrocytes within plasma, avoiding the influence of buffer, dilution, filters and filtration pressure. Tk, that was high in HD and partially corrected post-HD, was markedly increased in KT. ED is crucial for a correct oxygen carriage, since erythrocytes cross microcirculation where the diameter of capillaries is often less than 7 μm. Therefore, a decrease of ED causes: a) capillary hypertension and reduced oxygen delivery because of raised microcirculatory flow resistance, b) high endothelial shear stress, that is a key trigger in the cascade of hypertrophy, proliferation and fibrosis by means of production of vasoactive mediators, expression and redistribution of adhesion molecules, cytoskeleton changes and transcription of genes involved in processes of cell growth (i.e. c-myc, c-fos- c-jun) [17]. Thus, a low ED may contribute to the aggravation of cardiovascular risk in spite of good control of systemic blood pressure and of conventional and unconventional risk factors. Low ED could contribute to the progression of chronic graft dysfunction, a histological picture of interstitial fibrosis and tubular atrophy (IF/TA), result of immunological, toxic, vascular and infectious insults [16]. ID used in KT could possibly play a role in ED decreasing [2, 20]. We found no hemorheological differences in KT grouped for ID; furthermore, we found no difference between patients on CyA and on FK. The absence of hemorheological differences between the classes of ID in KT may be due to the small case series and to the rheological effects of other drugs.

In conclusion, HD patients showed marked hemorheological changes; this could contribute to support the high incidence of cardiovascular complications in these patients, involving large vessels (ηS₁, EAI), myocardial hypertrophy (τ₀), small vessels and microcirculation (ηS₂₀₀, Tk, G’). In KT most of the hemorheological parameters resulted comparable to HV. However, the reduced ED measured in KT could act as a detrimental injury in microcirculation, damaging the endothelium and, through its activation, leading to a progression of organ damage.