Regina Michelis

Salivary β2-Microglobulin Analysis in Chronic Kidney Disease and Hemodialyzed Patients

Background: β2-Microglobulin (β2m) is a major component in dialysis-related amyloidosis, a disabling disease affecting long-term dialysis patients. Methods: β2m and other components were analyzed in saliva and serum from 53 individuals in 4 subgroups: healthy normal controls, diabetes mellitus (DM), chronic kidney disease (CKD) and hemodialysis (HD) patients. Results: Median salivary and mean serum β2m concentrations were 78% higher in both saliva (p = 0.048) and serum (p = 0.047) in DM patients; 118% (p = 0.016) and 907% (p = 0.007) higher, respectively, in CKD patients, and 328% (p = 0.0001) and 2,710% (p = 0.001) higher, respectively, in HD patients, compared with healthy controls. The correlation analysis between salivary and serum β2m concentrations showed a low correlation rate in HD patients (r = –0.18), but was rather high in CKD patients (r = 0.50). Conclusions: Salivary analysis of β2m is a reliable method for evaluating serum β2m levels in CKD patients, and may help predict the potential for development of CKD-induced amyloidosis.

In-vivo oxidized albumin– a pro-inflammatory agent in hypoalbuminemia

Hypoalbuminemia of Hemodialysis (HD) patients is an independent cardiovascular risk factor, however, there is no mechanistic explanation between hypoalbuminemia and vascular injury. In the event of oxidative stress and inflammation to which HD patients are exposed, albumin is oxidized and undetected by common laboratory methods, rendering an apparent hypoalbuminemia. We wanted to show that these circulating modified oxidized albumin molecules cause direct vascular damage, mediating inflammation. Once these in-vivo albumin modifications were reduced in- vitro, the apparent hypoalbuminemia concomitantly with its inflammatory effects, were eliminated. Albumin modification profiles from 14 healthy controls (HC) and 14 HD patients were obtained by mass spectrometry (MS) analyses before and after reduction in- vitro, using redox agent 1,4 dithiothreitol (DTT). Their inflammatory effects were explored by exposing human umbilical endothelial cells (HUVEC) to all these forms of albumin. Albumin separated from hypoalbuminemic HD patients increased endothelial mRNA expression of cytokines and adhesion molecules, and augmented secretion of IL-6. This endothelial inflammatory state was almost fully reverted by exposing HUVEC to the in-vitro reduced HD albumin. MS profile of albumin modifications peaks was similar between HD and HC, but the intensities of the various peaks were significantly different. Abolishing the reversible oxidative modifications by DTT prevented endothelial injury and increased albumin levels. The irreversible modifications such as glycation and sulfonation show low intensities in HD albumin profiles and are nearly unobserved in HC. We showed, for the first time, a mechanistic link between hypoalbuminemia and the pro-inflammatory properties of in-vivo oxidized albumin, initiating vascular injury.

Unexpected Normal Colloid Osmotic Pressure in Clinical States with Low Serum Albumin

Background In clinical states associated with systemic oxidative stress (OS) and inflammation such as chronic kidney disease (CKD), oxidative modifications of serum albumin impair its quantification, resulting in apparent hypoalbuminemia. As the maintenance of oncotic pressure/colloid osmotic pressure (COP) is a major function of albumin, this study examined the impact of albumin oxidation on COP, both in-vivo and in-vitro. Methods Patients with proteinuria and patients on chronic hemodialysis (HD) with systemic inflammation and OS were enrolled. Blood samples were collected from 134 subjects: 32 healthy controls (HC), proteinuric patients with high (n = 17) and low (n = 31) systemic inflammation and from 54 patients on chronic hemodialysis (HD) with the highest levels of OS and inflammation. Results In-vitro oxidized albumin showed significantly higher COP values than non-oxidized albumin at identical albumin levels. In vivo, in hypoalbuminemic HD patients with the highest OS and inflammation, COP values were also higher than expected for the low albumin levels. The contribution to COP by other prevalent plasma proteins, such as fibrinogen and immunoglobulins was negligible. We imply that the calculation of COP based on albumin levels should be revisited in face of OS and inflammation. Hence, in hypoalbuminemic proteinuric patients with systemic OS and inflammation the assumption of low COP should be verified by its measurements.

Free circulating active elastase, contributes to chronic inflammation in hemodialysis patients

Atherosclerosis and cardiovascular complications are prevalent among patients undergoing chronic hemodialysis (HD). In this population, peripheral polymorphonuclear leukocytes (PMNLs) are primed, releasing proinflammatory mediators such as elastase. Elastase is normally inhibited by a specific inhibitor, avoiding undesirable degradation of cellular and extracellular components. This study tested the hypothesis that in states of noninfectious inflammation, elastase is released by PMNLs and acts in an uncontrolled manner to inflict vascular damage. Blood was collected from patients undergoing HD and healthy controls (HC). PMNL intracellular and surface expressions of elastase were determined by quantitative real-time PCR, Western blotting, and flow cytometry. The elastase activity was evaluated using a fluorescent substrate. The levels of serum α1-antitrypsin (α1-AT), the natural elastase inhibitor, were determined by Western blot. Free active elastase was elevated in HD sera, whereas the levels of α1-AT were decreased compared with HC. The levels of the intracellular elastase enzyme and its activity were lower in HD PMNLs despite similar expression levels of elastase mRNA. Elastase binding to PMNL cell surface was higher in HD compared with HC. The increased circulating levels of free active elastase released from primed HD PMNLs together with the higher cell surface-bound enzymes and the lower levels of α1-AT result in the higher elastase activity in HD sera. This exacerbated elastase activity could lead to the endothelial dysfunction, as hypothesized. In addition, it suggests that free circulating elastase can serve as a new biomarker and therapeutic target to reduce inflammation and vascular complications in patients on hemodialysis.

The relationship between oxidized serum albumin and blood pressure in hypoalbuminemic peritoneal dialysis patients

ABSTRACT Background: Oxidative stress produces molecular modifications of serum albumin that disturb its biological functions and interfere with its detection by the bromocresol green assay (BCG). Oxidative stress, inflammation, and hypoalbuminemia are common peritoneal dialysis (PD). This study aimed to evaluate the relationship between serum albumin, oxidized serum albumin (OSA), oncotic pressure, and blood pressure in hypoalbuminemic PD patients. Methods: Twenty-four PD patients with serum albumin levels <3.5 g/dl enrolled in the study. Data were compared between participants with the mean arterial pressure (MAP) <105 mmHg (n = 12) and MAP ≥ 105 mmHg (n = 12). Results: Serum albumin levels were ≤3.0 g/dl and similar in both groups (p = 0.298). The calculated OSA and oncotic pressure were significantly higher in patients with MAP ≥ 105 mmHg than in those with MAP < 105 mmHg. MAP was positively and marginally correlated with serum albumin levels (measured by BCG) (r = 0.34, p = 0.05), and positively and significantly correlated with the calculated OSA and oncotic pressure (r = 0.44, p = 0.015, r = 0.58, p = 0.002; respectively). The oncotic pressure was positively correlated with the calculated OSA (r = 0.47, p = 0.011). Conclusion: OSA, undetectable by the commonly used BCG, may contribute to higher blood pressure in hypoalbuminemic PD patients.

The impact of oxidized serum albumin on the oncotic pressure and hydration status of peritoneal dialysis patients

Objective Hypoalbuminemia, fluid overload (FO), and oxidative stress (OS) may be related to cardiovascular morbidity and mortality in peritoneal dialysis (PD) patients. OS produces molecular modifications of serum albumin that interfere with its quantification by the commonly used bromocresol green assay. This study evaluated the impact of oxidized serum albumin (OSA) on oncotic pressure (OP) and hydration status. Patients and methods Twenty-four stable hypoalbuminemic PD patients were enrolled in the study. After performing physical examination, assessment of the hydration status using a whole-body bioimpedance spectroscopy technique was performed, and blood samples were drawn for determination of OP, serum albumin levels, and OSA. Results Extracellular to total body water (E/TBW) ratio was higher in patients with FO ≥1.5 L with or without edema than in patients with FO <1.5 L (P≤0.043). E/TBW ratio was higher in patients with FO ≥1.5 L and edema compared to those with FO ≥1.5 L but without edema (P=0.004). OP was significantly higher in patients with FO ≥1.5 L and without edema compared to those with FO ≥1.5 L and with edema (P<0.001). Albumin-detection index (ADI) in patients with FO ≥1.5 L and without edema was similar to ADI in patients with FO <1.5 L (P=0.520). ADI was significantly lower in patients with FO ≥1.5 L and without edema compared to those with FO ≥1.5 L and edema (P=0.034). E/TBW ratio correlated positively with the ADI (r=0.60, P=0.001) and inversely with the OP (r=−0.54, P=0.002). Conclusion Overhydration may be clinically undetectable in PD patients. Assessing the hydration status and measuring the total serum albumin levels, including the oxidized fraction, should be considered in evaluating hydration status in PD patients.