Nocturnal Hemodialysis Improves Erythropoietin Responsiveness and Growth of Hematopoietic Stem Cells

Clinical Observations

We studied 16 stable patients with ESRD (age 47 ± 2 yr; nine men). Of the 16 patients, 13 were white, two were Asian, and one was Hispanic. Their ESRD vintage was 5.7 ± 1.3 yr. Their ESRD was due to glomerulonephritis (n = 7), polycystic kidney disease (n = 3), diabetes (n = 2), vasculitis (n = 1), thrombotic microangiopathy (n = 1), reflux nephropathy (n = 1), and calcineurin antagonist toxicity (n = 1).

The dialysis dosage received (Kt/V per session) increased significantly after conversion to NHD (from 1.27 ± 0.06 to 2.23 ± 0.09; P < 0.01), and the frequency of dialysis doubled (Table 1). In addition, parathyroid hormone (PTH) levels fell (from 49.0 ± 5.4 to 20.6 ± 6.2 pmol/L; P < 0.05), and plasma phosphate concentration was restored to normal (from 2.1 ± 0.2 to 1.2 ± 0.1 mmol/L; P < 0.01). Concomitantly, there was a fall in mean arterial BP from 123 ± 4 to 106 ± 2 mmHg (P < 0.05) with a decrease in vasoactive medication requirement from 2.5 to 0.5 medications per patient (P < 0.001). Specifically, five of the study cohort were prescribed angiotensin-converting enzyme inhibitors or angiotensin receptor blockers at baseline. After 2 mo of NHD, two patients remained on angiotensin-converting enzyme inhibitors. Plasma albumin concentrations, aspirin use, and iron dosing did not change before and after conversion to NHD.

After 2 mo of NHD, hemoglobin levels improved from 113 ± 3 to 125 ± 4 g/L (P = 0.03) without alterations in EPO requirements or iron status (Table 1, Figure 1). Of the 16 study patients, three had a decrease in hemoglobin concentration, whereas the remainder had an increase. The observed reductions in PTH levels were directly correlated with the change in hemoglobin concentrations (P = 0.52, P = 0.04).

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Figure 1.

Changes in hemoglobin concentration before and after conversion to nocturnal hemodialysis.

Cell Culture Studies

Paired plasma samples obtained from the same patient while on CHD and NHD were examined to determine their ability to support colony formation by BFU-E and CFU-GM obtained from normal donors. The frequency and size of colonies grown in culture from the same target cells was superior with 20% NHD plasma compared with 20% CHD plasma (BFU-E: 190 ± 54 versus 61 ± 30 colonies; P = 0.02 [normal 352 ± 76]; CFU-GM: 94 ± 25 versus 34 ± 16; P = 0.03 [normal 189 ± 11], respectively; Figure 2).

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Figure 2.

Representative blast-forming units. (A through C) Erythroid appearance under CHD (A), NHD (B) and normal (C) conditions.

Gene Profiling

We performed gene profiling studies on mononuclear cells obtained from the peripheral blood of patients with ESRD while on CHD and subsequently on NHD. In addition, we conducted the same studies on pooled hematopoietic colonies grown from normal target cells in the presence of plasma derived while on CHD or NHD.

The principal components analysis of the differential gene expression patterns revealed two distinct populations coinciding with their degree of uremic clearance. Peripheral blood obtained from patients on NHD showed differential gene expression profiles that revealed augmentation in genes responsible for HPC mobilization and growth and production of RBCs (Table 2, Figure 1). Of potential candidate genes, significant upregulations in matrix metalloproteinase 9 (MMP-9; 1.65-fold increase), platelet factor 4 (1.69-fold increase), and chemokine receptor 4 (CXCR4; 1.38 fold increase) were noted after 2 mo of NHD. In addition, genes responsible for the biosynthesis of hemoglobin were systematically upregulated (Table 2). Similar expression profiles were obtained from normal target cells cultured in the presence of NHD plasma in comparison with the respective CHD plasma samples. Data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and are accessible through Gene Expression Omnibus Series accession no. GSE11227.

Plasma PTH levels inversely correlated with CXCR4 expression levels (r = −0.9, P < 0.001). CXCR4 expression was associated with levels of MMP-9 (r = 0.7, P < 0.05) and hemoglobin β (r = 0.8, P < 0.05).

Clinical Protocol

NHD patients received HD at home for 6 to 8 h, five to six nights per week. Vascular access was achieved through either a long-term internal jugular catheter (Uldall Catheter; Cook Critical Care, Bloomington, IN) or an arteriovenous fistula. Dialysate flow rate of 350 ml/min and blood flow rate of 200 to 300 ml/min was used. F80 polysulfone dialyzers (Fresenius Medical Care, Lexington, MA) or Exceltra 120 dialyzers (Baxter, Chicago, IL) were used. CHD patients received HD for 4 h three times per week via similar vascular access. A blood flow rate of 400 ml/min, a dialysate flow rate of 500 to 750 ml/min, and F80 polysulfone dialyzers (Fresenius Medical Care) were used. Unfractionated heparin was used for anticoagulation on CHD and NHD.

Dialysis dosage per treatment was estimated by equilibrated Kt/V (eKt/V) as described by Daugirdas et al.²⁴: eKt/V = spKt/V − 0.6(spKt/V)/t + 0.03, spKt/V = single-pool Kt/V.²⁵ spKt/V was determined using blood URR.

Patients included consecutive eligible patients who were converted to NHD at the University Health Network. Patient demographic information such as age, gender, ethnicity, cause of ESRD, and comorbid conditions was prospectively collected into a computerized clinical database. Clinical assessment, including weight, height, and BP measurements, were performed at baseline and monthly after conversion to NHD. Biochemical and hematologic parameters (complete blood count, urea, creatinine, albumin, alkaline phosphatase, calcium, phosphate, and PTH) were obtained monthly during the same time intervals. Baseline studies were performed the morning after a CHD day (a minimum of 18 h after dialysis). To minimize circadian variation and replicate steady-state NHD conditions, we performed subsequent experiments at the same time of day (a minimum of 4 h after the regular NHD session).

Differential Gene Expression under CHD and NHD Conditions

Whole blood samples were obtained atraumatically through the clinical protocol already outlined at baseline (under CHD) and after 2 mo of exposure of NHD. The blood samples were processed immediately after withdrawal to maximize the fidelity of the gene expression data.

TRNA was isolated from blood samples using Trizol Reagent (Life Technologies/BRL, Grand Island, NY) following the manufacturer's protocol. The quality of TRNA was assessed using the Agilent 2100 Bioanalyzer (version A.02.01S1232; Agilent Technologies, Santa Clara, CA). Only RNA with the OD ratio of 1.99 to 2.00 at 260/280 was used for microarray analysis. Hybridizations were performed on the Human HG-U133_PLUS2 GeneChip (Affymetrix, Santa Clara, CA). Samples were prepared for hybridization according to standard Affymetrix instructions and performed at the Genomic Core Facility at the Hospital for Sick Children. Microarrays were performed in compliance with accepted Microarray Experimental Guidelines.²⁶

To monitor and compare differential gene expression in CHD and NHD samples, data obtained from GCOS (GeneChip Operating software) absolute analysis of all of the arrays were analyzed and clustered using GENESPRING 7.0 (Agilent Technologies). After filtering, one-way ANOVA (nonequal variance) was performed and differentially expressed genes were identified by hierarchical clustering. The same method was used to establish gene expression profiles from pooled hemopoietic colonies grown from normal peripheral blood stem cell donors in the presence of either CHD or NHD plasma obtained from the same patient.

Effect of Uremic Plasma on Normal HPCs

As described previously,²⁷ HPC-derived colonies from normal individuals were grown in semisolid cultures supported by normal heparinized human plasma and sources of growth factors. The latter included human recombinant growth factors IL-3 (50 ng), GM-CSF (50 ng), SCF (50 ng), and EPO (2 U) and 10% phytohemaglutinin-conditioned medium. The need for human plasma at a concentration of 20% provided the opportunity to compare the growth supporting effect of normal plasma with plasma derived from patients with ESRD while initially on CHD and subsequently on NHD. Thus, plasma of each patient on study was evaluated under both dialysis conditions. Normal peripheral HPCs served as targets. They were obtained from consenting healthy donors of G-CSF–mobilized hemopoietic allografts. The cultures were scored for the presence of BFU-E–derived erythroid burst and CFU-GM–derived granulocyte-macrophage colonies. Individual colonies were removed, and TRNA was prepared. Differential gene expression analysis was performed using methods outlined already.

Data Analysis

Descriptive data are presented as means ± SE. The primary outcome measure was the difference in HPC colonies among normal control subjects and CHD and NHD patients. Differentially expressed genes were identified by hierarchical clustering before and after conversion to NHD. Mann-Whitney U test was used for comparison of continuous variables between two groups. ANOVA was used for multiple comparisons of a continuous variable among three groups of patients. Spearman correlation was used to investigate potential associations between variables of interest. All statistical tests were two tailed with a P < 0.05 taken to indicate significance. SPSS 10 (SPSS Inc., Chicago, IL) was used for all statistical analyses.