Bioimpedance Resistance Indices and Cell Membrane Capacitance Used to Assess Disease Status and Cell Membrane Integrity in Children with Nephrotic Syndrome

Background Accumulation of extracellular water (ECW) is a major clinical manifestation of nephrotic syndrome (NS) in children. Bioimpedance spectroscopy (BIS) is a simple, noninvasive technique that reflects body water volumes. BIS can further measure cell membrane capacitance (CM), which may be altered in NS. The aims of the study were to explore how BIS measurements could reflect disease status in NS, while avoiding prediction equations which are often only validated in adult populations. Methods The study involved 8 children (2-10 years) with active NS (ANS group), 5 of which were also studied at NS remission (NSR group), as well as 38 healthy children of similar age (HC group). BIS measurements determined resistances RINF, RE, and RI (reflecting total body water, extracellular water, and intracellular water) and CM. Also resistance indices based on height (H) were considered, RI = H2/R. Results It was found that RE and RINF were significantly lower in the ANS group than in both NSR and HC groups (p < 0.001). Corresponding resistance indices were significantly higher in the ANS group than in the NSR (p < 0.01) and the HC (p < 0.05) groups, in accordance with elevated water volumes in NS patients. Indices of intracellular water were not significantly different between groups. CM was significantly lower in the ANS group than in NSR and HC groups (p < 0.05). Conclusion BIS could distinguish children with active NS from well-treated and healthy children. Studies with more children are warranted.


Introduction
Nephrotic syndrome (NS) is a kidney disease characterized by the massive leakage of proteins into the urine, with consequent hypoalbuminemia and oedema formation [1], increasing the risk of complications and prolonged hospitalization [2]. Clinicians typically use body weight as a measure of the volume status in NS patients. However, this approach does not provide information about the different body water compartments; furthermore, using body weight as an indicator of volume status is only reliable for time periods short enough that changes in adipose tissue and muscle mass are nonsignificant [3].
Availability of a simple, inexpensive, and harmless method for routine assessment of volume status could provide new and clinically useful information to clinicians in the treatment of children with NS. Bioelectrical impedance spectroscopy (BIS) may prove to be such a method. BIS is noninvasive, harmless, quick, simple, and an inexpensive method that can be performed with a portable instrument. These features make it suitable for routine use.
Bioelectrical impedance is the opposition (impedance) of the body tissue against the flow of electrical current. The bioimpedance depends on the body composition, especially the distribution of water and nonwater in the body. Scales claiming to measure not just your weight but also your 2 The Scientific World Journal Cell ＃ -２ ) ２ ％ Figure 1: Model representing the electrical properties of body tissue [21]. R E corresponds to the electrical resistance offered by the extracellular water (ECW) and likewise R I from the intracellular water (ICW). C M represents (at the body level) cell membrane capacitance of the double-layered cell membrane, i.e., the cell's ability to uphold a charge difference between the two sides of the membrane. The BIS device applies an alternating current (AC) and measures the combined impedance. Due to C M , the impedance will depend on the frequency of the electrical current, because highfrequency currents pass the cell membrane with less opposition than low-frequency currents. R E and R I are measured in Ohm (Ω), while C M is measured in nanofarad (nF).
body composition make use of bioimpedance; however, most of these devices measure only at a single frequency. BIS measures bioimpedance at a wide range of frequencies, giving more complete and more reliable information. BIS has recently been used to demonstrate increased volumes of extracellular water (ECW) in NS patients compared to controls [4]. As well as being able to provide information about the volume of the different body water compartments, BIS also estimates cell membrane capacitance (C M ), which has been suggested as the parameter directly linked to the cell membrane function [5]; low C M has been linked to disease in children [6]. To our knowledge, however, C M has not been studied before in relation to NS patients.
While bioimpedance-based approaches for quantitative estimation of water volumes exist, these methods are based on prediction equations, which are prone to bias and imprecision. When only a single frequency is measured, the prediction equations are typically simple and purely empirical [7]. This makes the equations sensitive to the choice of population (e.g., adults but not children) as well as the precision of the reference method. In the multiple-frequency approach of BIS, empirical data are incorporated into equations derived from a biophysical model of body water compartments, their volumes, and inherent electrical resistivity [5]. However, even equations involving analytical derivations will depend on their assumptions of, e.g., homogeneity of the body. Accordingly, such prediction equations have been found inaccurate and population specific [8][9][10] in paediatric populations.
An alternative approach to using prediction equations may be the use of BIS resistance parameters, either as the resistances (R) or as the so-called resistance indices (RI) [11][12][13]. The resistances are calculated directly from the measured BIS data, while the resistance indices are calculated from the resistances and the subject's height.
Briefly, to the BIS apparatus, the body can be represented by the electrical circuit shown in Figure 1. The measured parameters comprise the resistances R E , R I , and R INF , representing the electrical resistances of ECW, intracellular water (ICW), and total body water (TBW = ECW + ICW), respectively. For more details on BIS theory, see [5,14]. Given the resistances R E , R I , and R INF , the corresponding resistance indices RI E , RI I , and RI INF can be calculated, which (unlike the resistances) are roughly proportional to the water volumes [15].
Generally, there is a growing interest from the scientific community in the use of raw impedance data as indices of body water volume, especially in patients with altered body water distribution [16,17]. These indices may be less intuitive than absolute body water volumes in L, but avoid invoking the various assumptions that underpin the prediction equations.
The aims of the present study were to investigate how BIS measurements can reflect disease status in NS, including both resistances (R E , R I , and R INF ), corresponding resistance indices, and capacitance C M . For an overview of parameters and abbreviations, see Table 1.

Material and Methods
Standardized testing and reporting procedures were followed as far as possible [15,18]. The datasets produced during and/or analysed during the present study are available from the corresponding author on reasonable request.

Subjects.
Eight children (7 boys, 1 girl, age range: 2-10 years) with active NS (ANS group) were enrolled at the Department of Paediatrics and Adolescent Medicine, Aarhus University Hospital, Denmark. Inclusion criteria were the presence of NS defined by proteinuria >40 mg/m 2 /day, plasma albumin <25 g/L, oedema, and hyperlipidemia. Exclusion criteria were low plasma levels of C3-complement, postinfectious glomerulonephritis, and vasculitis such as Henoch-Schönlein nephritis of specific glomerulonephritis as, for example, dense deposit disease. Five of the ANS patients (ANS * group) were also restudied on remission (NSR group). Remission was defined as urinary dipstick negative for protein on three consecutive days. Blood samples, blood pressures, and impedance measurements were collected in the ANS patients before treatment with prednisolone and diuretics was initiated.
For comparison, impedance measurements were also made in 38 healthy control children (HC group) (23 boys, 15 girls, age range: 2-10 years). These controls were taken from a previously published dataset [20].

Ethics.
Informed consent was obtained from the subjects' parents or legal guardians before study enrolment. The study was performed in accordance with the Helsinki Declaration and was approved by the Central Denmark Region Committees on Health Research Ethics (case number: 1-10-72-17-12).

Patient Preparation.
Participants were not fasting but had refrained from intense physical exercise four hours prior to measurements and had been resting in the supine position for 5 min before measurement and remained at rest (no movement) during measurement. There were no restrictions on voiding. Participants remained clothed with only hands and feet uncovered with the body positioned with the arms and legs abducted at a 30-45 ∘ angle from the trunk. , which measures the impedance at 50 frequencies in the range from 5 to 1000 kHz. All measurements were performed in accordance with and earlier published paper [20]. Briefly, skin surface Ag-AgCl ECG-style gel electrodes were placed at wrists and ankle for whole-body measurements, and the skin was cleaned with alcohol (ethanol 75%) before the placement of electrodes. Measurements were performed with participants lying supine on a nonconductive surface (hospital bed/examination table) and abducted arms and legs. Intertwining of cables and similar sources of electrical interference was avoided.
Calibration of the device was tested every week with an electronic verification module (TS4201), supplied by the manufacturer (Xitron Technologies, San Diego, CA, USA).
All measurements were made at room temperature (21 ∘ to 25 ∘ C), between 08:30 and 15:30, by the same trained operator, and performed in triplicate with electrodes remaining in place between measurements. The total mean measurement time was 7 min, covering patient preparation and BIS measurements.

BIS Data
Analysis. The quality of the impedance data was checked by visual inspection and statistical analysis of the Cole plots, using the ImpediMed SFB7 Multi-Frequency Analysis software (Version 5.4.0.3, Brisbane QLD, Australia) as described previously [22].
The precision of triplicate measurements made in each NS patient was based on the electrical parameters R E and R I [15,18] and expressed by the coefficient of variation in percent (CV% = SD/mean • 100%).
The average CV% showed to be low for R E (0.5%) and R I (1.5%) in the ANS patients (n = 8 triplicate measurements) and for R E (0.2%) and R I (0.4%) in the NSR patients (n = 5 triplicate measurements).
Resistance varies with the length and cross-sectional area of the conductor. For current passing through a body, neither path length nor cross-sectional area is well-defined. However, a resistance index (RI) can be calculated, which takes body size into account and is roughly proportional to the water volume [15]:  Data are means ± SD; for group abbreviations, see Table 1; BMI: body mass index. 10.6 7.9 * Patients in the subgroup ANS * , which were restudied at remission, see Table 1. † Hypertension defined as blood pressure above the 95% percentile for high and gender; ‡ eGFR estimated by the Schwartz formula [19]. Reference ranges: P-Albumin (37-48), P-Sodium (137-145), P-potassium (3.5-4.6), and B-hemoglobin (6.5-8.9). clothes to within 0.1 kg. Height was measured without shoes, to the nearest 0.5 cm using a stadiometer. All measurements were made in duplicate with mean values used.

Biochemistry and Blood Pressure.
Resting venous blood samples and blood pressures (Carescape V100 Monitor, GE Healthcare, USA) were collected in the ANS patients before commencing medical treatment. Blood chemistry was performed by trained biomedical laboratory scientists in an accredited hospital laboratory.

Statistics.
Results were presented as mean ± standard deviation (SD), after test for normality, using Q-Q plots and statistical tests (Shapiro-Wilk and Kolmogorov-Smirnow) [23].
A paired two-tailed Student's t-test was applied to determine differences in impedance data between patients with active NS and at remission (ANS * vs NSR). An unpaired twotailed t-test was used to compare impedance data between patients with active NS with controls (ANS vs HC) and at remission with controls (NSR vs HC).
Statistical significance was set at a p-value < 0.05. All statistical tests and graphical illustrations were prepared using the statistical software MedCalc 5 (Version 17.9.7, Medcalc Software, Ostend, Belgium). Table 2 summarizes the characteristics of all the subjects enrolled. When comparing the body weight of the groups, no significant changes were observed (p > 0.05). Table 3 details the clinical data for the patients. All patients had normal or near normal renal function, and four of eight patients were hypertensive at admission.

Patient Characteristics.
Impedance measurements were only possible in five patients during remission because of repeated relapse in two of the patients and transition to another hospital of one patient.
One ANS patient was notable for being an apparent outlier for weight ( Figure 2, right panel, and Figure 3, right panel); this patient was at the 100-percentile of weight for his age, using data from the 'WHO child growth standards' [24]. Table 4 and corresponding p-values in Table 5. Resistance indices are presented as a function of age and weight in Figure 2. Cell membrane capacitance is likewise presented in Figure 3. A summary of the details is given in the following.

Bioimpedance Parameters and Indices. Data are presented in
Overall, the results showed no significant differences between the children in submission (NSR group) compared to the healthy control children (HC group). Regarding ICW, the corresponding BIS parameter (R I ) and resistance index (RI I ) showed no significant differences between sick children (ANS group and ANS * subgroup) and healthy controls (HC). See Table 5.
The mean values of R E and R INF were significantly lower for the sick children (ANS and ANS * ) than for the children The Scientific World Journal 5   60  55  50  45  40  35  30  25  20  15  10  5  0   70  65  60  55  50  45  40  35  30  25  20  15  10  5  0   12  11  10  9  8  7  6  5  4  3  2  1  0  70  65  60  55  50  45  40  35  30  25  20  15 Table 1 for group descriptions; dashed lines are 95% confidence intervals for regression lines based on the HC data. 6 The Scientific World Journal  Table 1 for group descriptions; dashed lines are 95% confidence intervals for regression lines based on the HC data. in remission (NSR) or being healthy (HC). In numbers, the difference between ANS and HC groups was 369.2 Ω (-45%) for R E and -212.6 (-37%) for R INF (Table 4). Correspondingly, the resistance indices RI E and RI INF (but not RI I ) were significantly higher for the sick children than those in remission or being healthy (ANS * vs NSR and ANS vs HC, respectively). In numbers, the difference between ANS and HC groups was +14.9 cm 2 /Ω (+75%) for RI E and +14.7 cm 2 /Ω (+51%) for RI INF (Table 4). These elevated values of RI E and RI INF (but not RI I ) are graphically visible in both panels of Figure 2.
Cell membrane capacitance (C M ) was also found to be significantly lower for the sick children (ANS and ANS * ) than for the children in remission (NSR) or healthy (HC). The graphical plots (Figure 3) reveal that only some of the individual ANS patients had lower C M than the HC children.

Discussion
To the authors' knowledge, this paper demonstrated for the first time the relation between changes in disease status and cell membrane capacitance in paediatric patients with NS.
These results were obtained using impedance spectroscopy, which allows the calculation of resistance at the optimal frequencies for ECW (f = 0) and TBW (f = ∞).
In the present study, BIS clearly indicated a higher ECW in the ANS patients compared to both the controls (HC group) and patients at remission (NSR group). Importantly, these findings accord with observations made in previous studies in NS patients [4,25], where absolute fluid volumes were estimated using predictive methods.
As described in the Introduction, the use of BIS-modelled resistances avoids the assumptions inherent in derived equations for predicting fluid volumes. R E represents the resistance of the ECW alone [26][27][28][29] while R E and R I have been used as indices of changes in ECW and ICW in neonates [11]. R E and R I are furthermore well-accepted parameters for characterizing material and biological properties, e.g., cell size, density, and the constituents of the extracellular and intracellular matrix [13,30]. In our study, significantly lower R E values were observed in the ANS patients compared to the HC. However, R I values were the same as those in the healthy control cohort, indicating that the size of the intracellular water compartment is unchanged in the acute phase of NS, while the extracellular compartment alone is affected by the disease.
In contrast to the resistance values, resistance indices (RIs) standardize the values according to the subject's height. RIs have been shown to be an accurate reflection of absolute water volumes in children [31][32][33]. For the ANS patients, we observed that RI E and RI INF values were higher compared to the HC, and comparable with the HC for RI I . Again, it appears that only extracellular, not the intracellular, compartment is affected.
Consideration of basic BIS parameters can include electrical parameters other than resistance. Specifically, charge differences over cell membranes results in a capacitive effect [5]. In whole-body impedance analysis, cell membrane capacitance (C M ) is a parameter measured at body level and represents an average of all membranes and tissue interfaces along the conductive path. Through cell death or cell destruction, The Scientific World Journal 7 the cell membrane loses its high capacitive properties [5], and upon general cell death, ECW expands and C M decreases [5].
Even though there is no reason to suspect cell death or cell destruction in connection with NS, our results indicates that the cell membrane can be affected by the disease, despite the fact that ICW seems unaffected. This may indicate that there are components of the disease that affect the membrane negatively, e.g., inflammation and immune system activation [34]. Intriguingly, the pathogenesis of NS has previously been explained by a loss of negative charge in the glomerular basement membrane leading to heavy proteinuria. It is, however, unlikely that such changes would be discernible in a measurement of whole-body C M . This raises the possibility that these alterations in cell membrane function extend beyond the glomerular [35], but that possibility remains to be investigated more fully. Significant (p < 0.001) changes in C M compared to healthy controls have also been observed in children suffering from sickle cell disease (SCD) [36].

Limitations.
Some limitations to the study should be acknowledged. Only a small number of patients were available for study. This is a consequence of the low incidence of the disease with only around 2 new cases per 100,000 [37]. This carries the risk that the studied patients were not representative of NS patients in general; this is, however, considered unlikely since they exhibit the comparable clinical characteristics to other patients in the clinic. Even though significant differences between the sexes in body composition even when adjusted by height have been found in studies in healthy children [38], our sample was not large enough to conduct such an analysis.
Impedance measurements were performed as a wristankle (whole-body) analysis. This arrangement does not make it possible to determine how the individual segments or discrete regions of the body contribute to the overall impedance with respect to NS, i.e., to identify in which of the segments/part where the greatest impedance changes occur. To obtain such information, it is necessary to perform focal or segmental measurements [15]. Normative data for segmental BIS in children aged 1-18 years are available for comparison [39], but it is unknown whether such an approach would be of value in NS patients.

Conclusion
This study shows how simple resistance indices could be used to assess changes in disease status in children with NS. This approach avoids invoking assumptions underpinning prediction of absolute volumes. More widely, such indices may prove to be useful screening tools for detection of fluid compartment imbalance in a clinical setting where the volume status of at-risk patients needs to be evaluated. In addition, it was observed that changes in C M reflect disease status, suggesting that the capacitive properties of the cell membranes are affected by the disease. For further understanding of C M in NS patients, studies are required with data specific for the different stages of NS that can be correlated with tissue pathology and, e.g., sex-specific differences.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
The study was performed in accordance with the Helsinki Declaration and was approved by the Central Denmark Region Committees on Health Research Ethics (case number: 1-10-72-17-12).

Consent
Informed consent was obtained from the subjects' parents or legal guardians before study enrolment.

Conflicts of Interest
Author Leigh C. Ward provides consultancy services to ImpediMed Ltd. ImpediMed Ltd. had no involvement in the preparation of this manuscript. Other authors have no conflicts of interest to declare concerning this work.