Hemorheologic alterations in peritoneal dialysis

1 Introduction

End Stage Renal Disease (ESRD) condition has been expanding steadily in the past years; according to the United States Renal Data System (USRDS) annual data report of 2015 [29] the ESRD prevalence continues to rise with its consequent social and economic burden. Although mortality for ESRD patients has declined in the past two decades, it remains substantially higher than in the general population, the major contributor to excess mortality being cardiovascular (CV) disease [3, 29]. Patients with chronic uremia have multiple conditions predisposing to disorders of coronary perfusion and to atherosclerosis [19]. Traditional risk factors as hypertension, dyslipidemia, diabetes mellitus, physical inactivity are extensively present in ESRD population and result usually aggravated by uremia. Moreover, a number of uremia-related CV risk factors have been identified, such as hemodynamic overload, anemia, increased oxidant stress, hypoalbuminemia, inadequate dialysis, divalent ion abnormalities, metabolic acidosis, hypo/hyperkalemia [19]. Hemodialysis (HD) and peritoneal dialysis (PD) are considered equivalent treatment options for ESRD, since observational data suggest that there is no difference in survival between the two modalities [17, 33]. Notwithstanding, a recent study [31] showed that after five years of follow-up in patients below 65 years PD yielded superior survival rates compared to HD, thus encouraging its use as initial dialysis modality in ESRD patients. Nevertheless, and possibly related to the use of glucose-based solutions for peritoneal dialysis, increase in body weight and body fat mass, lipid profile disorders, hyperglycaemia and hyperinsulinemia more frequently develop in PD patients, and the risk of these patients for developing cardio-metabolic syndrome is higher than that of HD patients [11].

Hemorheologic parameters are emerging as an important biomarker for vascular dysfunction [32]. Mechanical interaction between blood and vessels has a crucial role in the release of endothelium-derived mediators (NO and endothelin) and subsequent vascular remodelling; an increased and prolonged alteration of blood viscosity and/or erythrocytes deformability may play a pathogenic role in the activation of endothelium, initiating inflammation, alteration of lipid metabolism, and progression of atherosclerotic vascular disease [2]. In the cohort of the Edinburgh Artery Study, high blood viscosity was associated with a significant rise in the hazard ratio for incident myocardial infarction in conjunction with other biomarkers in multivariate analysis, after adjustment for CV risk factors and history of CV disease [28]; however, authors concluded that the predictive ability of single biomarkers was modest and their clinical utility remained uncertain. Recently, few papers [7, 22] have explored the possibility of determining the magnitude of microcirculatory alteration in the kidney with the use of contrast enhanced ultrasonography; this technique may be able to provide important and early diagnostic information on microcirculatory status in kidney disease. Hemorheologic alterations have been extensively described in HD patients. Importantly, Martinez et al. showed an increase in plasma viscosity and erythrocyte aggregability in HD patients, although erythrocyte deformability was reported as normal [15]; Brimble et al. described higher blood viscosity values at all shear rates in HD patients, both diabetic and non-diabetic [1]; more recently, Reinhart et al reported that the passage through a hemodialysis filter induced erythrocytes shape changes, increased the hematocrit, whole blood and plasma viscosity, decreased RBC aggregation, and affected platelet aggregation [21]. Contrasting with the myriad of reports regarding HD, literature provides very few data regarding hemorheologic characteristics of PD patients. In a small observational study, Feriani et al. [8] observed that patients with ESRD treated with PD had higher plasma viscosity (despite lower plasma protein concentration) and whole blood viscosity compared to HD patients and controls; a controlled trial [16] showed that hemorheological abnormalities were more marked in patients on PD than in those on HD and were not improved by folate supplementation. Microvascular damage has been previously recognized in PD patients [23], although the causes remain incompletely understood.

One problem with PD is its time-limitation, with rates of technique failure at 5 years being substantially more elevated than in HD, partly due to loss of ultrafiltration function of the peritoneum [4]; moreover, long-term PD patients appear to be prone to a rare but devastating disease named encapsulating peritoneal sclerosis, whose pathogenesis remains still uncertain [18]. Unraveling the microvascular “dilemma” of PD may help tailoring this treatment to the appropriate patient and shed light on its dysfunctions and complications.

2 Methods

Our aim is to characterize the hemorheologic profile of PD patients comparing it with our data regarding HD patients and healthy control volunteers (HV). Our primary endpoint was to determine differences in erythrocyte deformability (estimated through Taylor’s factor, see below) between PD and HD patients. All participants were asked to sign informed consent before obtaining blood specimens for routine and hemorheologic measures. This research complies with the requirement for ethical publication in Clinical Hemorheology and Microcirculation as published in Clin Hemorheol Microcirc. 2010;44(1):1-2.

We measured hemorheologic parameters of 49 PD patients, treated both with continuous ambulatory PD and with automated nocturnal PD. We compared data obtained with those in our possess [10] regarding 90 patients undergoing intermittent HD (where blood samples were collected before the second dialysis session of the week, pre-HD) and 47 HV. Demographic characteristics are summarized in Table 1. Baseline characteristics of dialysis patients are shown in Table 2.

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 [27]. 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 [25]. Erythrocyte deformability (ED) was evaluated as Taylor factor [26] (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.

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 (interquartile range); 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).

3 Results

In the following exposition results will be expressed as medians (interquartile range). ηP was normal in PD, and lower than in HD (PD vs pre-HD: 1.4 (1.34–1.44) vs 1.6 (1.37–1.73) mPa.s); see Fig. 1. Plasma levels of albumin and other protein fractions in PD and HD groups are expressed in Table 2. ηS₁ resulted lower in PD when compared to HD (PD vs pre-HD: 14.17 (11.05–18.33) vs 23.14 (16.89–34.97) mPa.s), but it was higher than in HV (data shown in Fig. 2). EAI resulted normal in PD (PD vs pre-HD: 2.61 (2.31–3.85) vs 5.60 (4.35–7.71)); see Fig. 2. τ₀ was also normal in PD (PD vs pre-HD: 0.09 (0.07–0.13) vs 0.17 (0.13–0.23) mPa); see Fig. 2. ηS₂₀₀ was altered in PD with values being markedly higher than in HV (PD vs HV: 4.58 (4.11–5.02) vs 3.63 (3.06–3.99) mPa.s), and comparable to those found pre-HD (data shown in Fig. 3). Tk was markedly and significantly higher in PD than in HD (PD vs pre-HD: 0.9 (0.85–0.95) vs 0.81 (0.78–0.86); p < 0.0001) and HV (HV: 0.77 (0.70–0.83)); see Fig. 4. Analysis of viscous-elastic blood behaviour confirmed higher G’ in PD than in HD and HV (Linear Regression, 95% confidence interval for slopes: HV 0.007445 to 0.01497, pre-HD 0.01595 to 0.02663, PD 0.03286 to 0.04168; for slopes difference p < 0,001); see Fig. 4.

4 Discussion

Our results confirm an alteration of hemorheological parameters in PD, although with important differences with respect to HD patients. Generally, PD patients showed an improvement of parameters related to macro-circulation (ηP, ηS_1, EAI, τ₀) when compared to HD, while microcirculatory function resulted severely impaired (as expressed by ηS₂₀₀, Tk, G’).

In contrast with previous reports [8, 16], ηP resulted normal in PD, comparable to values found in HV. Although α-globulin fractions of total plasma proteins were higher in PD than in HD patients, the concomitant and significant decrease in γ-globulins may be responsible for the reduction in ηP; reciprocal interactions between protein fractions may also play a role. ηS₁ (highly influenced by erythrocyte aggregation and plasma viscosity) and EAI both resulted lower in PD than in HD; the reduction in erythrocytes aggregability expressed by these two parameters theoretically allows the maintenance of a physiologic laminar flow in low blood areas (such as venules), reducing thrombotic risk [6]. τ₀ in PD patients was low and comparable to values found in HV. 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, chronically contributing to myocardial hypertrophy. ηS₂₀₀ values in PD patients resulted higher than those of HV and comparable to HD patients; an elevation in ηS₂₀₀ in dialysis patients could be ascribed to a low ED, since at high velocity rate erythrocytes are completely disaggregated and blood viscosity is mainly determined by ED. 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 (data not shown) [10], was significantly increased in PD patients; data on G’ confirmed this finding, showing a “stiffer” viscous-elastic behavior for PD patients’ blood (see Fig. 4). A reduced ED in PD patients had been previously reported, and was not corrected despite fish-oil treatment [12]; a possible etiology is represented by the exposure to high-glucose dialysis fluids in PD, with accumulation of glucose degradation products (GDPs) and subsequent generation of reactive oxygen species [13]. We found no differences in blood glucose levels between the two groups of patients (Table 2), but single glucose measurement does not reflects overall glucose exposure and GDPs at peritoneal level. It is worth to mention that in our analysis PD patients had higher levels of PTH than HD patients; it is unlikely for this alteration alone to explain the worse ED in PD patients, since serum calcium and phosphorous were within the normal range (see Table 2). Proper ED is crucial in microcirculation where erythrocytes are forced to cross capillaries with a diameter that is often less than 7μm; in case of a reduction in ED, a pathologic shear stress is imposed to the endothelium, with consequent secretion of vasoactive mediators and vascular remodeling factors that lead to an acceleration in the atherosclerotic process, common in many ESRD patients. Vlahu et al. [30] reported a substantial loss in glycocalyx barrier properties (as estimated by analysis of the dynamic variations of erythrocyte column width in the sublingual microcirculation) in both HD and PD patients; although analysis of ED was not included in that study, a pathogenic role for impaired ED in the disruption of glycocalyx is plausible, and it could represent the first step in endothelial activation leading to the aggressive vascular pathology present in this group of patients. Effects of these alterations at systemic level obviously translate into the extremely high prevalence of CV disease and complications in ESRD patients.

If atherosclerosis is a systemic vessel disease, it may also influence the function of peritoneal capillaries; thus, the over mentioned hemorheologic alterations will also affect peritoneal microcirculation. In this regard, it is important to examine late complications of PD. The mail limitation for long-term PD is the inability of peritoneal membrane to perform an adequate transport of water and solutes; the most frequent alteration is the water transport failure or loss of ultrafiltration function (UF) [20], combined with an increase in small solute transport. UF represents the most diffuse cause for technique failure in PD and need for a switch to other renal replacement therapies [20]. Although loss of UF has been extensively attributed to the exposure to high-glucose dialysis fluids, impaired ED (in turn, possible consequence of the chronic exposure to GDPs) might act as a detrimental factor in a multi-hit process that ultimately leads to peritoneal fibrosis. Histopathological study of peritoneum in PD patients has shown, beyond the extensive sub-mesothelial fibrosis, striking alteration in the vascular system, such as reduplication of sub-endothelial basal membrane and neoangiogenesis [20, 24]. Peritoneal vascular alteration are presumed to be driven by the up-regulation of Vascular Endothelial Growth Factor (VEGF), which in turn provokes endothelial hyper-permeability and proliferation; since VEGF mRNA expression is induced by exposure to low-oxygen tension through the Hypoxia-Inducible Factor (HIF-1) mediation [5, 9], low ED in PD patients is likely to play an important role, causing microvascular congestion and tissue hypoxia. Encapsulating peritoneal sclerosis is a rare but devastating complication of long-term PD, poorly responsive to treatment and whose pathogenesis is still partly unexplained. Although simple peritoneal sclerosis linked to the chronic exposure to GDPs appears to be the necessary milieu where the disease can manifest, a second hit (acute cessation of PD, frequent peritonitis, transplantation, possible genetic predisposition) is usually necessary. Impaired microvascular function and endothelial damage may contribute to the pathogenesis of peritoneal encapsulating sclerosis, as the process of Epithelial-to-Mesenchimal Transition preceding fibrosis and ultimately sclerosis can occur under the influence of a number of pro-inflammatory and pro-fibrotic cytokines [18] usually produced in an ischemic milieu.

5 Conclusions

We found hemorheologic alterations in PD patients, with substantial differences with respect to HD patients. Although ηP was normal and both ηS₁ and erythrocyte aggregability resulted substantially lower than in HD, PD patients showed profound alteration in microcirculatory flow, as expressed by high values of ηS₂₀₀ and strikingly worse ED than in HD. Impairment in ED may act as a risk factor for accelerated atherosclerosis and precipitate cardiovascular events; moreover, it may have a detrimental effect in the peritoneal microcirculation promoting endothelial activation with subsequent fibrosis, leading to peritoneal membrane malfunctioning.