The systemic repercussions of the progression of chronic kidney disease (CKD) can compromise the functionality of subjects undergoing hemodialysis (HD), since changes in the respiratory and musculoskeletal systems are common in this population.1–5 Pulmonary congestion is the result of excess fluid, reduced oncotic pressure and greater permeability of the blood-air barrier due to an increase in uremic toxins and inflammatory mediator concentrations, and may lower lung compliance and predispose individuals to restrictive ventilatory defects.3,6 As such, this condition can alter the mechanics of breathing and exacerbate clinical symptoms, compromising the activities of daily living (ADL) of subjects with kidney disease.

Pulmonary congestion is a strong predictor of mortality and cardiovascular events in CKD.6,7 Additionally, previous research has shown that moderate and severe degrees of pulmonary congestion are associated with reduced physical function in subjects undergoing HD and peritoneal dialysis.8,9 However, these studies used self-reporting questionnaires and pedometers, which are known to be inaccurate and limited in terms of assessing ADL. Moreover, the population studied consisted of subjects with moderate and severe degrees of pulmonary congestion, making it difficult to draw conclusions about the functional aspects related to those with milder forms of the condition.

Thus, it is important to investigate the repercussions of mild pulmonary congestion in terms of the respiratory mechanics and functionality of these individuals. The aim of this study was to assess the influence of mild pulmonary congestion on diaphragmatic mobility (DM) and ADL in HD subjects, as well as compare ADL behavior on days with and without HD. Besides that, to provide a better understanding of the possible mechanisms involved in this process by performing an experimental research with adenine-induced CKD animal model.

Respiratory muscle strength was measured using a digital manometer (MVD300®; Globalmed, Porto Alegre, Brazil), according to the guidelines of the Brazilian Pulmonology and Thoracic Society.18 The largest value obtained during maximal inspiratory (MIP) and expiratory pressure (MEP) maneuvers was considered for analysis, expressed as an absolute value and percentage of the predicted normal value.17

Symptoms were functionally classified (NYHA scale) in order to place subjects in one of four categories based on how much they were limited during physical activity: I – no symptoms during ordinary physical activity, with limitations similar to those expected in healthy individuals; II – mild symptoms during ordinary activity; III – marked symptoms even during less-than-ordinary activity; IV – symptoms present at rest.19

Experimental study

The experimental research was submitted to the Ethics Committee on Animal Use (CEUA) of the Universidade Federal de Santa Catarina (no. 9256210519) and to the Universidade do Estado de Santa Catarina (no. 2306201018). All the animals received humane care in compliance with EU Directive 2010/63/EU and Animal Research: Reporting In Vivo Experiments (ARRIVE), which provides guidelines for animal experiments. Twenty male Swiss mice (30–40g), young adults, aged 8–10 weeks, were maintained in controlled conditions for temperature (22±2°C), humidity (70–75%) and dark/light cycle (12h; lights on at 07:00 am). The diet consisted of water and ration ad libitum. The animals were randomized in 2 groups: Control (C, n=10) and Chronic kidney disease (CKD, n=10). The sequence of CKD induction, testing period and euthanasia are illustrated in Fig. 1.

Fig. 1.

Experimental design.

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CKD was induced by adding 0.2% adenine (SIGMA®) (i.e. 0.2g adenine/100g feed) to the mice diet for four weeks, following the model established by Ali et al. (2013).20 The Control group animals, in turn, received the conventional ration (BioBase®, BioTec line, Chapecó, Santa Catarina, Brazil).

All the animals were adapted to the treadmill (Advanced 2, Athletic®, Santa Catarina, Brazil) for three days, performing 10min of activity at a speed of 0.2km/h. Twenty-four hours after adaptation, all the mice were submitted to the MPCT. The MPCT, adapted from Vieira et al. (2007)21 was performed with five minutes of heating at 0.2km/h, followed by an increase in velocity of 0.1km/h each 2.5min, until animal exhaustion, observed when mice could no longer run, even after 10 small mechanical stimuli. The travelled distance (km) for each animal was recorded.

Open Field Test (OFT) was performed to evaluate the exploratory locomotor activity of mice. Each mouse was placed alone in an open field arena (40×340m) and animal behavior was recorded during five minutes by a digital camera. The images were analyzed by Any-maze software in a blinded fashion. We evaluated the number of rearings (exploratory movement in which the animal stands).22

Animals were put under anesthesia with isoflurane 1–2%, through a face mask and positioned in supine. A triplex scanning ultrasound device (Logiq 3, General Electric Company, Milwaukee, Wisconsin, 53201, United States of America), with a 6–10MHz linear array probe, was used to assess for the presence of pulmonary congestion.23

After performing the thoracic USG, the animals were anesthetized with xylazine and ketamine. A surgical incision was made in the midline of the animals’ abdomen, and a blood sample from the abdominal aorta artery was collected in heparinized syringes with approximately 1mL. Samples were centrifuged (4000rpm, at 4°C, for 15min) and the resulting serum aliquoted and stored at −80°C for further analysis of plasma creatinine and urea concentrations by spectrophotometry using commercial kits. After blood collection, animals were euthanized by arterial section.

Kidney tissue samples were homogenized and the supernatant stored at −80°C. Total protein was measured in the supernatant using the Bradford method, and levels of TNF-α (R&D Systems – Minneapolis, MN, United States of America) were analyzed, according to manufacturer's instructions. Tissue cytokine levels were estimated by interpolation from a standard curve using colorimetric measurements at 450nm (540nm wavelength correction) on an ELISA plate reader (Berthold Technologies – Apollo 8 – LB 912, KG, Germany). The results of the tissue samples were expressed as picogram per milligram (pg/mg).

Of the 30 subjects with CKD studied, 56.6% were women and approximately 50% were classified as eutrophic based on their BMI (Table 1). All the participants were under conventional HD.

Participants exhibited a decline in percentages of predicted values for the spirometric variables (FVC, FEV1 and MVV), and reduced inspiratory muscle strength when compared to predicted values. No subjects displayed obstructive disorders in the lung function assessment. The mean of DM was 59.76mm and a mean of 7.13 lung comet-tails were observed. Half of the participants were classified as category I on the NYHA scale, demonstrating that their symptoms did not limit their daily activities. However, the other 50% of the sample reported limiting symptoms even when presenting less severe pulmonary congestion (Table 2).

There was a moderate correlation between pulmonary congestion and DM (r=−.48; P=.008). Simple linear regression revealed that mild pulmonary congestion reduced DM by 26.1% (R2=.261; P=.004), indicating a 2.23mm decrease in DM for every unit increase in lung comet-tails (Fig. 2A). A moderate correlation was also observed for pulmonary congestion and walking time (r=−.49; P=.006). Simple linear regression revealed that mild pulmonary congestion decreased walking time by 20% in HD subjects (R2=.200; P=.01), indicating a 1.54-min decline for every unit increase in lung comet-tails (Fig. 2B).

Fig. 2.

Correlation of pulmonary congestion with the DM and walking time. DM: diaphragmatic mobility.

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Regarding the ADL behavior, no statistical difference was observed between the variables time spent sitting (328.18±128.85min vs. 322.98±111.42min; P=.86), time spent lying down (295.3±142.52min vs. 274.15±130.6min; P=.44) and sedentary time (623.49±46.8min vs. 597.13±76.38min; P=.10) on days with and without HD, respectively. The statistically significant differences were observed only for the variables standing time, walking time, active time and number of steps taken (Table 3).

Parallel to these findings, the experimental study also showed corresponding results, since there was a significative impairment of creatinine clearance in CKD group, as described in Table 4.

The CKD group also showed a significant increase of TNF-α in both kidney (P=.037) and lung (P=.02) tissues, compared to the Control group (Fig. 3).

Fig. 3.

Kidney and lung levels of TNF-α. C: control group; CKD: chronic kidney disease group.

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In order to ratify the lung impairment, we also evaluated the histological pattern of adenine-induced CKD mice, and our findings on a qualitative increase in collagen content in lungs of CKD group, as shown in Fig. 4.

Fig. 4.

Representative photomicrographs of lung tissue stained with Picro-Sirius red at 400× magnification. C: control group; CKD: chronic kidney disease group.

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The physical capacity of the animals was assessed by the maximum travelled distance (km) during the MPCT. After adenine induced CKD, the animals in the CKD group showed a significant decrease in the travelled distance (P=.034) when compared to the Control group (Fig. 5A). The exploratory and locomotor activity were evaluated by the OFT, the absolute number of rearings over five minutes was recorded as an indirect measure of functionality. This assessment is related to the animal's ability to support its own weight on its legs (Fig. 5B). After adenine intake, the CKD group had a statistically significant reduction in the number of rearings when compared to the Control group (P=.01).

Fig. 5.

Effect of adenine induced CKD on maximum travelled distance during MPCT and absolute number of rearings. MPCT: maximum physical capability test; C: control group; CKD: chronic kidney disease group.

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Finally, thoracic USG was able to identify the presence of B-lines in the lungs of CKD mice, a strong indication of CKD-associated pulmonary congestion (Fig. 6).

Fig. 6.

Thoracic USG of animals assessed. CKD: chronic kidney disease.

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The main finding of our study was that even low levels of fluid buildup in the lungs can compromise DM and ADL performance in HD individuals. In the experimental context, although it is not possible to evaluate the grade of pulmonary congestion through USG in small animals, it was possible to identify the presence of B-lines, and based on this it may be suggested that its presence may have played a decisive role on poor physical performance and exploratory behavior of adenine-induced CKD mice.

In our sample, 26% of DM was influenced for the mild pulmonary congestion. Although there was no apparent change in DM in the subjects studied here, there are no reference values in the literature for this outcome, making it difficult to confirm. However, studies that assessed DM in healthy individuals during deep breathing via USG found average values close to 60mm,25,26 similar to the 59.76mm recorded here. Although diaphragm dysfunction was not present, mild pulmonary congestion was capable of compromising diaphragm movement, likely due to the negative effects of fluid buildup, such as a decline in lung compliance, volumes and capacities caused by restrictive ventilatory defects.3,6 Thus, because the participants exhibited only mild pulmonary congestion, we hypothesize that more severe forms of the condition may exert an even greater influence due to increased lung restriction. Applying clinical and physical therapy techniques to reduce excess fluid accumulation would therefore be a relevant strategy in this population. Since our study is the first to investigate the effects of pulmonary congestion on DM, further research is needed to better understand changes in the respiratory mechanics in CKD.

Our results suggest that ADL may be compromised in CKD subjects with mild pulmonary congestion, because walking time was influenced in 20% by fluid buildup in the lungs. In parallel, MPCT was performed by mice in this study, as a measure of functionality, and the animals presented a significant reduction in the maximum travelled distance. Another measure that can be associated with ADL in humans is the number of rearings performed by the animals at OFT, which also showed a significant reduction. Similar results were reported by Enia et al. (2013)8 in individuals with moderate and severe pulmonary congestion, whereby an increase of 10 lung comet-tails raised the probability of worse physical performance by 23%. In light of these findings, our results reinforce the fact that even mild pulmonary congestion can compromise the functionality of HD subjects. As such, introducing interventions aimed at minimizing the effects of pulmonary congestion would allow for greater improvement in functional capacity in these individuals, even for milder forms of the condition.

The ADL behavior of these subjects on dialysis and non-dialysis days was also compared. A statistical difference was observed for the variables standing, walking and active time, and number of steps taken, whereby individuals exhibited an increase in active behavior on non-dialysis days. Several authors have used questionnaires and equipment to estimate energy expenditure or number of steps taken per day and observed sedentary behavior and inactivity in this population when compared to healthy individuals.27–32 Additionally, our results corroborate those reported by Gomes et al (2015),33 who found that subjects took fewer steps, walked less, and spent less time standing and more lying down on non-dialysis days, largely due to the period of inactivity during dialysis. There are reports in the literature that these subjects are 24% less active on dialysis days, with the treatment itself responsible for this decine.27 These findings indicate that dialysis subjects tend to be sedentary, particularly on treatment days, making it important to provide physical training programs during HD in order to improve their activity levels.

With respect to pulmonary function, the results obtained corroborate those reported in the literature.1,34,35 Approximately 67% of participants (n=20) displayed some form of restrictive ventilatory defect in the pulmonary function test, demonstrating that CKD subjects tend to experience limited ventilation due to excess fluid accumulation and respiratory muscle dysfunction.1,36 Moreover, our sample also exhibited a decline in FEV1 percentage, but no obstructive lung disorders. Previous studies have suggested that reduced FEV1 in these individuals is the result of fluid overload near the small airways, hampering breathing.34,37

In regard to respiratory muscle strength, mean MIP was below predicted normal values. Participants also showed a 50% decline in predicted normal MVV values, a spirometric variable related to muscle endurance. Thus, our individuals exhibited a reduction in both inspiratory muscle strength and respiratory muscle endurance. Previous research reports that decreased muscle strength in CKD is the result of factors such as systemic inflammation, metabolic acidosis, hormonal changes and nutrient deficiencies, which occur as the disease progresses and culminate in reduced muscle mass, strength and endurance, condition known as sarcopenia.5,36 The experimental data supports this information, since levels of TNF-α were elevated both in kidney and lung tissues, reflecting this state of chronic inflammation and probably influencing on the reduction in respiratory muscle strength and endurance. Previous studies with the elderly and subjects with chronic obstructive pulmonary disease have found a reduction on diaphragmatic thickness and MIP when these conditions were associated with sarcopenia.38,39 Based on this, sarcopenia in CKD, in addition to affecting the peripheral musculature, could compromise the respiratory musculature, contributing to a reduction in lung capacity and the emergence of dyspnea in this population.1,3

The symptoms of half of the participants studied here were categorized as class I on the NYHA scale and 20% fell under classes III and IV. This suggests that a small portion of individuals with mild pulmonary congestion may already experience significant symptoms that limit their daily activities. Salerno, Parraga and Mcintyre (2017)3 reported that dyspnea may occur in CKD subjects due to the emergence of pulmonary congestion, which reduces lung compliance, restricts lung parenchyma and hampers gas exchange across the blood-air barrier. This, combined with impaired respiratory muscles, can further exacerbate the disease.40

Our study was innovative in that it investigates the influence of pulmonary congestion on functional outcomes and respiratory mechanics in a group of HD individuals typically excluded from research for exhibiting a less severe form of the condition. The intention of running an experimental set in parallel was to provide background and some extra information to justify the findings and make them more robust. A limitation in the translation of data was not being able to ensure the mild level of pulmonary congestion in the animals, only to prove the presence of it. The animals were not at the end-stage of renal disease, so they did not need HD, and we were not able to perform pulmonary mechanics measurements, like DM. Regarding clinical data, residual diuresis and interdialytic weight gain are variables that can influence the function of the cardiac muscle and the pulmonary fluid build-up in our population. Thus, not measuring these variables can be considered a limitation in our study. The lack of knowledge about the prevalence of sarcopenia can be considered as another limitation, making it difficult to understand the negative effects of this dysfunction on the respiratory muscles of CKD subjects. Another limitation of the study is that we did not use fluoroscopy, which is capable of evaluating DM in real time. However, it is important to underscore that indirect DM measurement by craniocaudal displacement of the left branch of the portal vein has proved to be a practical, valid and reproducible technique.11,12 Failure to measure excursion of the left hemidiaphragm is another limitation, but a previous study indicated no statistical differences between right and left DM measurements,41 making it easier to measure this outcome in clinical practice.

In spite of these limitations, the results of the present study demonstrate the importance of assessing and monitoring subjects in HD even when they exhibit only mild pulmonary congestion. Although the pathophysiology of CKD differs from that of chronic lung and heart diseases, its progression has similar systemic repercussions that can compromise functional capacity and increase sedentary behavior and cardiovascular-related mortality in this population. In this sense and as previous studies have reported better outcomes in CKD individuals undergoing aerobic exercise, peripheral and respiratory muscle training,42,43 structured rehabilitation programs need to be encouraged and implemented during HD.

The present findings allow us to infer that mild pulmonary congestion reduced DM and walking time in subjects undergoing HD. The ADL behavior differed in this population on days with and without HD, with individuals being less active on dialysis days. Furthermore, the experimental model implies that the presence of pulmonary congestion and inflammation may play a decisive role in the low physical and exploratory performance of CKD mice.

The study was funded by the Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC) (no. 2017TR645).

The authors have disclosed no conflicts of interest.