Beta-glucans in advanced CKD: role in endotoxaemia and inflammation.

BACKGROUND/AIMS: (1-3)-ß-D glucans (BG) are cellular components of yeasts and fungi. Elevated blood levels may be an adjunct in diagnosing invasive fungal infection, though can be high in dialysis patients without fungaemia. BG can also induce false positive signals in endotoxin detection assays (Limulus Amoebocyte Lysate [LAL] assay). We explored the relationship between BG levels, renal impairment, endotoxaemia and inflammation. METHODS: We measured serum BG levels, markers of inflammation and blood endotoxin levels in 20 controls, 20 with stages 1-3 chronic kidney disease (CKD), 20 with stages 4-5 CKD, 15 on peritoneal dialysis (PD) and 60 on haemodialysis (HD). Another 30 patients were studied before and after HD initiation. RESULTS: BG levels increased with advancing CKD, being highest in HD patients, 22% of whom had elevated levels (> 80 pg/ml). Levels increased significantly following HD initiation. Levels also correlated positively with CRP, TNFα, IL-6 levels, independently of CKD stage. Blood endotoxin was detectable by LAL assays in 10-53% of the CKD cohort, being most prevalent in the HD group, and correlating positively with BG levels. Adding BG blocking agent to the assay reduced endotoxin detection confining it to only 5% of HD patients. Levels of inflammatory markers were higher in those with detectable endotoxin - whether false- or true positives. CONCLUSION: BG levels increased with decreasing renal function, being highest in dialysis patients. High BG levels were associated with false positive blood endotoxin signals, and with markers of inflammation, independently of CKD stage. The cause for high BG levels is unknown but could reflect increased gut permeability and altered mononuclear phagocytic system function.

Diabetes confers in vitro calcific potential on serum which associates with in vivo vascular calcification.

Although vascular calcification (VC) is prevalent in Type 2 diabetes mellitus (T2DM), underlying mechanisms remain unclear. Neither is it known whether T2DM confers calcific potential (CP) on serum, enabling it to induce VC outside the disease milieu. We, therefore, investigated the CP of serum from controls and subjects with T2DM with and without in vivo VC. Samples from 20 healthy controls and 44 age- and sex-matched patients with T2DM with modification of diet in renal disease estimated glomerular filtration rate (MDRD-4 eGFR) > 60 ml·min-1 were analysed for CP using rat aortic smooth muscle cells in vitro CT scans of femoral arteries identified individuals with in vivo calcification. Serum from subjects with T2DM revealed significantly greater CP than controls. This was further enhanced in the presence of in vivo VC. Addition of ß-glycerophosphate (ß-GP) plus CaCl2 increased the CP of T2DM serum but not of controls. Along with age, CP was an independent predictor of the presence of VC. In receiver operator curve (ROC) analysis, CP was a significant predictor of femoral arterial VC (C-statistic 0.70: P=0.009). The distribution of CP was bimodal around a cutoff of 100 nmoles of Ca2+ protein mg-1, with a higher proportion of Type 2 diabetes subjects with in vivo calcification (T2DM+) sera above the cutoff value. This group also showed elevated levels of osteoprotegerin (OPG) and matrix Gla protein (MGP). Diabetes confers CP on the serum which is enhanced by the presence of in vivo VC. The CP acquired may be dependent on levels of OPG and MGP. These findings may be clinically relevant for early identification of individuals at risk of VC and for informing therapeutic strategies.

The Effect of Intra-Dialytic Exercise on Inflammation and Blood Endotoxin Levels.

BACKGROUND: In healthy individuals, an acute inflammatory response occurs after intense exercise due to gut ischaemia and intestinal bacterial endotoxin translocation into the bloodstream. This process maybe exacerbated in patients who exercise during dialysis due to large volume shifts experienced by many during haemodialysis (HD). The acute effect of intra-dialytic exercise on blood endotoxins and inflammation is not known. METHOD: The effect of intra-dialytic exercise on blood endotoxin and inflammation was investigated in 10 patients and compared with resting haemodialysis. Blood was measured for endotoxin and inflammatory biomarkers before and after dialysis. RESULT: With the exception of one sample, all samples tested negative for endotoxin. Intra-dialytic exercise attenuated the rise of interleukin-6, tumour necrosis factor-α and high-sensitivity C-reactive protein after the HD procedure. CONCLUSION: Intra-dialytic exercise was not associated with an observable rise in blood endotoxin, although it may ameliorate the inflammatory effects of the HD procedure. Larger studies are needed to confirm this finding.

Human uraemic serum displays calcific potential in vitro that increases with advancing chronic kidney disease.

Vascular calcification (VC) strongly correlates with declining renal function and contributes to the high morbidity and mortality of patients with CKD (chronic kidney disease). It is closely regulated by circulating factors but little is known about the capacity of serum from patients to induce calcification outside the disease setting, which we now define as the calcific potential of serum. We have therefore examined the ability of serum from age- and sex-matched subjects with and without advancing CKD to induce calcification of cultured SMCs (smooth muscle cells). Samples from patients with CKD induced significant calcification compared with controls. More importantly, samples from patients on haemodialysis induced significantly higher calcification than those with moderate or advanced CKD. The calcification induced by the latter two but not those on haemodialysis could be enhanced with calcium chloride and ß-GP (ß-glycerophosphate). A positive correlation was evident between measured serum creatinine, phosphate, PTH (parathyroid hormone), OPG (osteoprotegerin) and the degree of calcification in vitro. eGFR (estimated glomerular filtration rate), DBP (diastolic blood pressure), haemoglobin and serum albumin correlated negatively. Stepwise multivariate analysis of log-transformed calcific potential data highlighted serum creatinine, albumin and OPG as significant predictors, explaining approximately 50% of the variation. Thus, other regulators, either not investigated or as yet unidentified, may contribute to the calcification potential of serum in vitro. Furthermore, uraemic serum can induce graded calcification outside of the disease milieu that reflects the degree of kidney impairment in vivo. These findings could have important clinical relevance in terms of developing novel diagnostic and/or therapeutic strategies for subjects with CKD.

Uremic serum-induced calcification of human aortic smooth muscle cells is a regulated process involving Klotho and RUNX2.

Vascular calcification (VC) is common in subjects with chronic kidney disease (CKD) and is associated with increased cardiovascular risk. It is an active process involving transdifferentiation of arterial smooth muscle cells (SMCs) into osteogenic phenotype. We investigated the ability of serum from CKD subjects to induce calcification in human SMCs in vitro (calcific potential of sera: CP), and associated changes in expression of Runt-related transcription factor 2 (RUNX2), SM22α, and Klotho. Sera from subjects with CKD (18 stage 3, 17 stage 4/5, and 29 stage 5D) and 20 controls were added to human cultured SMCs and CP quantified. The CP of CKD sera was greater (P<0.01) than that of controls, though not influenced by CKD stage. Modification of diet in renal disease estimated glomerular filtration rate (MDRD-4 eGFR) (P<0.001), serum phosphate (P=0.042), receptor activator of nuclear factor κappa-B ligand (RANKL) (P=0.001), parathyroid hormone (PTH) (P=0.014), and high-density lipoprotein (HDL)/cholesterol ratio (P=0.026) were independent predictors of CP accounting for 45% of variation. Adding calcification buffer (CB: calcium chloride [7 mM] and ß-glycerophosphate [7 mM]) increased the CP of control sera to approximate that of CKD sera. CP of CKD sera was unchanged. CKD sera increased RUNX2 expression (P<0.01) in human SMCs and decreased SM22α expression (P<0.05). Co-incubating control but not CKD serum with CB further increased RUNX2 expression (P<0.01). Both SM22α and Klotho expression decreased significantly (P<0.01) in the presence of CKD serum, and were virtually abolished with stage 5D sera. These findings support active regulation by CKD serum of in vitro VC by induction of RUNX2 and suppression of SM22α and Klotho.

Is Endotoxemia in Stable Hemodialysis Patients an Artefact? Limitations of the Limulus Amebocyte Lysate Assay and Role of (1→3)-ß-D Glucan.

BACKGROUND: Elevated blood endotoxin levels are frequently reported in the dialysis population and are strongly linked with inflammation, a major predictor of mortality. Virtually all studies have employed the Limulus Amoebocyte Lysate (LAL) assay to detect endotoxin. However this assay is not endotoxin-specific and can be activated by (1→3)-ß-glucan (BG), a component of fungal cell walls leading to false positive signals. Very few studies have taken account of this. We examined the influence of BG-based activation of the LAL assay on the detection of endotoxemia in this setting. METHOD: We measured plasma endotoxin levels in 50 hemodialysis patients with and without the use of BG-blocking buffers. These buffers inhibit BG activation of the LAL assay to ensure that any signal detected is endotoxin-specific. Blood samples were measured for BG, interleukin-6 (IL-6), tumor necrosis factor-alfa (TNF-α) to examine the association between endotoxin signals, BG and inflammation. RESULTS: Endotoxin signals were detected in 50% of patients. On repeat measurement with a BG-blocking buffer, all detected endotoxin signals were extinguished. No patient had detectable endotoxemia. Plasma BG levels were significantly elevated in 58% of patients and were higher in those with detectable endotoxin signals using the LAL assay without BG-blocking buffers (78vs.54pg/mL;p<0.001). Endotoxin signal and BG levels did not correlate with levels of TNF-α or IL-6. CONCLUSION: Use of the LAL assay for blood endotoxin detection in dialysis patients has its limitations due to high blood BG. Endotoxemia frequently reported in non-infected hemodialysis patients may be artefactual due to BG interference.