Introduction:Cell-free DNA is an emerging marker of allograft injury, yet its role in the immediate phase of ischemia-reperfusion injury remains incompletely understood. Methods:In this prospective cohort of 127 kidney transplant recipients (86 deceased donors, 41 living donors), intraoperative plasma samples were collected systemically pre-implantation and from the allograft vein postreperfusion. Nuclear and mitochondrial cell-free DNA were quantified, alongside subset assessments of neutrophil extracellular trap markers and soluble C5b-9 as a marker of thromboinflammation. An in vitro necrosis model of human proximal tubular cells evaluated the concordance between cell injury and C5b-9 generation. Results:An immediate and sustained release of nuclear, but not mitochondrial, cell-free DNA was observed post-reperfusion, predominantly in deceased donor kidneys. This release corresponded with cold ischemic time and delayed graft function while showing temporal correlations with soluble C5b-9, indicating that cell-free DNA release parallels thromboinflammatory activation upon reperfusion. In vitro, C5b-9 generation occurred on necrotic, but not viable, tubular cells, supporting the relationship between cell injury and thromboinflammation. Neutrophil extracellular trap markers did not consistently correlate with early cell-free DNA release. Conclusion:Immediate nuclear cellfree DNA release upon reperfusion reflects intragraft injury linked to downstream thromboinflammatory activation, underscoring the impact of early ischemia-reperfusion injury.

BACKGROUND:Ischemia/reperfusion injury is an unavoidable consequence of kidney transplantation, yet the characteristics of the immediate immune response after reperfusion and its impact on transplant outcomes remain poorly characterized in the clinical setting. METHODS:We conducted a cohort study including 63 kidney transplant recipients (26 living-donor, 37 deceased-donor) with an extended 4-y follow-up to characterize early postreperfusion inflammatory dynamics and their association with transplant outcomes. Using high-throughput proteomics, we profiled 92 inflammatory markers in the early reperfusion stage. Intraoperative blood samples were collected systemically at baseline and from the transplant vein at 1, 10, and 30 min postreperfusion. RESULTS:Our analysis revealed a pronounced early immune response on reperfusion, with distinct inflammatory trajectories between living- and deceased-donor kidney allografts. Living-donor allografts showed proteomic patterns suggestive of a regulatory response, whereas deceased-donor allografts exhibited patterns associated with a cell injury-related response. Notably, interleukin-33 and hepatocyte growth factor were associated with delayed graft function, whereas hepatocyte growth factor also correlated with long-term allograft dysfunction. CONCLUSIONS:These findings underscore the potential of assessing early postreperfusion inflammation to improve clinical risk stratification and guide future biomarker validation efforts.

Ultrafiltration (UF) insufficiency in peritoneal dialysis (PD) often leads to transfer to hemodialysis. Therefore, strategies to prolong time on therapy are needed. The use of a combined crystalloid and colloid solution, also called bimodal PD, can help improve UF. We propose a method for reconstituting bimodal solutions. Three bimodal PD solutions, with respectively 100 ml (solution 100), 150 ml (solution 150) and 200 ml (solution 200) of 30

BACKGROUND:Water retention, ultrafiltration insufficiency, and metabolic complications due to abnormally high glucose concentrations are still common problems in patients treated with peritoneal dialysis. Phloretin, a nonselective inhibitor of facilitative glucose transporter channels (GLUT), has shown to improve water transport and lower glucose absorption in experimental peritoneal dialysis. However, the dose-response relationship remains unknown, and we therefore performed a dose-response study to elucidate the pharmacodynamic properties of intra-peritoneal phloretin therapy. METHODS:Experimental peritoneal dialysis was performed in fifty healthy Sprague-Dawley rats, using glucose-based dialysis fluid containing five different concentrations of phloretin. We utilized radiolabeled 18F-deoxyglucose (18-FDG) to determine the plasma-to-dialysate transport. The data was then analyzed to determine the dose-response relationship of phloretin according to the Hill-model equation. RESULTS:Intraperitoneal phloretin therapy followed a dose-response relationship where higher concentrations of phloretin lowered the diffusion capacity of 18-FDG and conventional glucose, while enhancing ultrafiltration. Phloretin showed high potency for water removal and diffusion outcomes, requiring low concentrations to achieve substantial effects. CONCLUSIONS:Intraperitoneal phloretin therapy followed a distinct dose-response relationship, showing high potency in improving ultrafiltration and reducing glucose absorption in experimental PD. These findings support the therapeutic potential of GLUT-inhibitors like phloretin and support future clinical studies to evaluate efficacy and optimal dosing in patients undergoing PD.

Peritoneal dialysis (PD) is becoming more popular as a result of a rising interest in home dialysis, lower intrusion in social life and longer preservation of residual kidney function. However, PD has several important drawbacks: small solute clearance is relatively low compared to hemodialysis and technique survival is limited. Application of continuous flow, sorbent-based dialysate regeneration and novel glucose-sparing PD solutions are some solutions proposed to address the limitations of PD. To optimize and personalize current and novel PD therapies, patient peritoneal characteristics interacting with PD techniques need to be studied together and separately as they interplay. However, considering the multitude of parameters, it would be difficult, expensive, and time consuming to optimize all parameter settings only with the help of clinical trials. Mathematical modelling is an exciting tool to dissect these interacting processes and comprehend PD techniques better at a patient specific level. In this review, we look at the history of computational PD models, explore the many ways a computational PD model can be constructed and review the various existing PD models that can be used to optimize and personalize PD treatment.

Computational models of peritoneal dialysis (PD) are increasingly useful for optimizing treatment in patients with kidney disease requiring dialysis (KDRD). However, although several mathematical models have been developed in the past few decades, a direct comparison of the models’ accuracy with respect to predicting in vivo data is needed to further create robust personalized models. Here, we used a dataset obtained in a previous in vivo experimental model of PD in pigs (23 sessions of 4 h 2 L dwells in four pigs) and humans (20 sessions in 20 patients) to compare six computational models of PD: the Graff model (UGM), the three-pore model (TPM), the Garred model (GM), and the Waniewski model (WM), as well as two variations of these (UGM-18, SWM). We conducted this comparison to predict the dialysate concentrations of key uremic toxins and electrolytes (four in humans) throughout a 4 h dwell. The model predictions can provide insight into inter-individual differences in ultrafiltration, which are critical for tailoring PD regimens in KDRD. While TPM offered improved physiological reality, its computational cost suggests a trade-off between model complexity and clinical applicability for real-time or portable kidney support systems. In future applications, such models could provide adaptive PD regimens for tailored care based on patient-specific toxin kinetics and fluid dynamics.

The recently discovered selective glomerular hypofiltration syndromes have increased interest in the actual elimination of molecules in the human kidney. In the present study, a novel human model was introduced to directly measure the single-pass renal elimination of molecules of increasing size. Plasma concentrations of urea, creatinine, C-peptide, insulin, pro-BNP, beta 2-microglobulin, cystatin C, troponin-T, orosomucoid, albumin, and IgG were analysed in arterial and renal venous blood from 45 patients undergoing Transcatheter Aortic Valve Implantation (TAVI). The renal elimination ratio (RER) was calculated as the arteriovenous concentration difference divided by the arterial concentration. Estimated glomerular filtration rate (eGFR) was calculated by the CKD-EPI equations for both creatinine and cystatin C. Creatinine (0.11 kDa) showed the highest RER (21.0 +/- 6.3%). With increasing molecular size, the RER gradually decreased, where the RER of cystatin C (13 kDa) was 14.4 +/- 5.3% and troponin-T (36 kDa) was 11.3 +/- 4.6%. The renal elimination threshold was found between 36 and 44 kDa as the RER of orosomucoid (44 kDa) was -0.2 +/- 4.7%. The RER of creatinine and cystatin C showed a significant and moderate positive linear relationship with eGFR (r = 0.48 and 0.40). In conclusion, a novel human model was employed to demonstrate a decline in renal elimination with increasing molecular size. Moreover, RERs of creatinine and cystatin C were found to correlate with eGFR, suggesting the potential of this model to study selective glomerular hypofiltration syndromes.

Background Volume overload is highly prevalent among patients treated with peritoneal dialysis (PD), contributes to hypertension, and is associated with an increased risk of cardiovascular events and death in this population. As a result, optimizing peritoneal ultrafiltration (UF) is a key component of high-quality dialysis prescription. Osmotic conductance to glucose (OCG) reflects the water transport properties of the peritoneum, but measuring it requires an accurate quantification of UF, which is often difficult to obtain because of variability in catheter patency and peritoneal residual volume. Methods In this study, we derived a new mathematical model for estimating UF during PD, on the basis of sodium sieving, using a single measure of dialysate sodium concentration. The model was validated experimentally in a rat model of PD, using dialysis fluid with two different sodium concentrations (125 and 134 mmol/L) and three glucose strengths (1.5%, 2.3%, and 4.25%). Then, the same model was tested in a cohort of PD patients to predict UF. Results In experimental and clinical conditions, the sodium-based estimation of UF rate correlated with UF rate measurements on the basis of volumetry and albumin dilution, with a R-2=0.35 and R-2=0.76, respectively. UF on the basis of sodium sieving was also successfully used to calculate OCG in the clinical cohort, with a Pearson r of 0.77. Conclusions Using the novel mathematical models in this study, the sodium dip can be used to accurately estimate OCG, and therefore, it is a promising measurement method for future clinical use.

Introduction Larger fill volumes in peritoneal dialysis (PD) typically improve small solute clearance and water removal, and vice versa—but the relationship between intraperitoneal volume and the capacities for solute and water transport in PD has been little studied. Here, it is proposed that this relative relationship is described by a simple ratio (Volumenew/Volumeold)2/3 up to a critical break-point volume, beyond which further volume increase is less beneficial in terms of solute and water removal. Method To scrutinize this hypothesis, experiments were conducted in a rat model of PD alongside a retrospective analysis of data from a prior clinical study. Rats underwent PD with either three consecutive fills of 8 + 8 + 8 mL (n = 10) or 12 + 12 + 12 mL (n = 10), with 45-minute dwell time intervals. This approach yielded 60 estimations of water and solute transport, characterized by osmotic conductance to glucose and solute diffusion capacities, respectively. Results Comparative analysis of the predictive efficacy of the two models—the simple ratio versus the break-point model—was performed using Monte Carlo cross-validation. The break-point model emerged as a superior predictor for both water and solute transfer, demonstrating its capability to characterize both experimental data from rats and clinical data from patients. Conclusion The present analysis indicates that relatively simple calculations can be used to approximate clinical effects on solute and water removal when prescribing a lower or higher fill volume to patients with PD.

Background Peritoneal dialysis (PD) is commonly performed using either intermittent or tidal exchanges, whereas other exchange techniques such as continuous flow PD are little used. Previous research indicated that stirring the intra-peritoneal dialysate markedly increases small solute clearances. Here, we tested the hypothesis that stirring of the dialysate increases small solute clearances by using a novel exchange technique where the dialysate is pulsed back and forth during the treatment without addition of fresh fluid.Methods PD was performed in anesthetized Sprague-Dawley rats with either no pulsations (20 mL fill volume), 2 mL (10%) pulses (21 mL fill volume), or 5 mL (25%) pulses (22.5 mL fill volume) utilizing a pulse flow rate of 5 mL/min. The higher fill volume for the pulsed treatments compensates for the fact that the average intra-peritoneal volume would otherwise be lower in pulsed treatments. Water and solute transport were closely monitored during the treatment.Results Net ultrafiltration decreased significantly during pulsed PD with the 25% pulse volume. The 60 min sodium dip was unaltered, whereas the fluid absorption rate was increased for the 25% group. Solute clearances did not significantly differ between groups, except for a slightly lower calcium clearance in the 25% group.Conclusion Our data indicate that stirring the dialysate using pulsed exchanges does not provide any advantage compared to conventional exchange techniques. In contrast, pulsed treatments had slightly lower ultrafiltration and small solute transport. The present findings may have implications regarding the choice of tidal volume in automated PD, favoring smaller tidal volumes.

Background Local and systemic side effects of glucose remain major limitations of peritoneal dialysis (PD). Glucose transport during PD is thought to occur via inter-endothelial pathways, but recent results show that phloretin, a general blocker of facilitative glucose channels (glucose transporters [GLUTs]), markedly reduced glucose diffusion capacity indicating that some glucose may be transferred via facilitative glucose channels (GLUTs). Whether such transport mainly occurs into (absorption), or across (trans-cellular) peritoneal cells is as yet unresolved. Methods Here we sought to elucidate whether diffusion of radiolabeled 18F-deoxyglucose ([18F]-DG) in the opposite direction (plasma → dialysate) is also affected by GLUT inhibition. During GLUT inhibition, such transport may either be increased or unaltered (favors absorption hypothesis) or decreased (favors transcellular hypothesis). Effects on the transport of solutes other than [18F]-DG (or glucose) during GLUT inhibition indicate effects on paracellular transport (between cells) rather than via GLUTs. Results GLUT inhibition using phloretin markedly reduced [18F]-DG diffusion capacity, improved ultrafiltration (UF) rates and enhanced the sodium dip. No other solutes were significantly affected with the exception of urea and bicarbonate. Conclusion The present results indicate that part of glucose is transported via the transcellular route across cells in the peritoneal membrane. Regardless of the channel(s) involved, inhibitors of facilitative GLUTs may be promising agents to improve UF efficacy in patients treated with PD.

BackgroundVariation in residual volume between peritoneal dialysis dwells creates uncertainty in ultrafiltration determination, dialysis efficiency, and poses a risk of overfill if the residual volume is large. Measuring the dilution of a marker molecule during fluid fill offers a convenient approach, however, estimation accuracy depends on the choice of dilution marker. We here evaluate the feasibility of creatinine and urea as dilution markers compared to albumin-based residual volumes and three-pore model estimations.MethodThis clinical, retrospective analysis comprises 56 residual volume estimations from 20 individuals, based on the dilution of pre-fill dialysate creatinine, urea and albumin concentrations during the dialysis fluid fill phase. Outcomes were compared individually. Bias induced by ultrafiltration, marker molecule mass-transfer and influence of fluid glucose contents was quantified using the three-pore model. Linear regression established conversion factors enabling conversion between the various marker molecules.ResultsCreatinine-based calculations overestimated residual volumes by 115 mL (IQR 89-149) in 1.5% dwells and 252 mL (IQR 179-313) in 4.25% glucose dwells. In hypertonic dwells, ultrafiltration was 52 mL (IQR 38-66), while intraperitoneal creatinine mass increased by 67% during fluid fill, being the leading cause of overestimation. Albumin-based volumes conformed strongly with three-pore model estimates. Correction factors effectively enabled marker molecule interchangeability.ConclusionsMass-transfer of low molecular weight marker molecules is associated with residual volume overestimation. However, by applying correction factors, creatinine and urea dilution can still provide reasonable estimates, particularly when the purpose is to exclude the presence of a very large residual volume.

Data from the original article "Relationship between fill volume and transport in peritoneal dialysis" by Carl Öberg, comprising experimental data from a rat model. Twenty Sprague-Dawley rats, half with 8+8+8 mL and half with 12+12+12 mL consecutive fill volumes, undergo peritoneal dialysis. Briefly, experiments start with a 45-minute dwell (with either 8 mL or 12 mL fill volume), followed by an additional fill (utilizing the same fill volume) and another 45-minute dwell period, and a final third fill and dwell. Dialysate sampling occurred at 15, 30, and 45 minutes for each dwell period.

A healthy and functional peritoneal membrane is key to achieving sufficient ultrafiltration and restoring fluid balance, a major component of high-quality prescription in patients treated with peritoneal dialysis (PD). Variability in membrane function at the start of PD or changes over time on treatment influence dialysis prescription and outcomes, and dysfunction of the peritoneal membrane contributes to fluid overload and associated complications. In this review, we summarize the current knowledge about the structure, function, and pathophysiology of the peritoneal membrane with a focus on clinical implications for patient-centered care. We also discuss the molecular and genetic mechanisms of solute and water transport across the peritoneal membrane, including the role of aquaporin water channels in crystalloid versus colloid osmosis; why and how to assess membrane function using peritoneal equilibration tests; the etiologies of membrane dysfunction and their specific management; and the effect of genetic variation on membrane function and outcomes in patients treated with PD. This review also identifies the gaps in current knowledge and perspectives for future research to improve our understanding of the peritoneal membrane and, ultimately, the care of patients treated with PD.

Conventional dialyzer membranes typically comprise of unevenly distributed polydisperse, tortuous, rough pores, embedded in relatively thick approximate to 20-50 mu m polymer layers wherein separation occurs via size exclusion as well as differences in diffusivity of the permeating species. However, transport in such polymeric pores is increasingly hindered as the molecule size approaches the pore dimension, resulting in significant retention of undesirable middle molecules (>= 15-60 kDa) and uremic toxins. Enhanced removal of middle molecules is usually accompanied by high albumin loss (approximate to 66 kDa) causing hypoalbuminemia. Here, the scalable bottom-up fabrication of wafer-scale carbon nanotube (CNT) membranes with highly aligned, low-friction, straight-channels/capillaries and narrow pore-diameter distributions (approximate to 0.5-4.5 nm) is demonstrated, to overcome persistent challenges in hemofiltration/hemodialysis. Using fluorescein isothiocyanate (FITC)-Ficoll 70 and albumin in phosphate buffered saline (PBS) as well as in bovine blood plasma, it is shown that CNT membranes can allow for significantly higher hydraulic permeability (more than an order of magnitude when normalized to pore area) than commercial high-flux hemofiltration/hemodialysis membranes (HF 400), as well as greatly enhance removal of middle molecules while maintaining comparable albumin retention. These findings are rationalized via an N-pore transport model that highlights the critical role of molecular flexing and deformation during size-selective transport within nanoscale confinements of the CNTs. The unique transport characteristics of CNTs coupled with size-exclusion and wafer-scale fabrication offer transformative advances for hemofiltration, and the obtained insight into molecular transport can aid advancements in several other bio-systems/applications beyond hemofiltration/hemodialysis.

Local and systemic side-effects of glucose remain major limitations of peritoneal dialysis (PD). Glucose transport during PD is thought to occur via inter-endothelial pathways, but recent data indicated that some glucose is transferred via facilitative glucose channels. Here we used BAY-876, a potent and highly selective blocker of facilitative glucose channel 1 (GLUT1), in an experimental rat model of PD using either 1.5% or 2.3% glucose fluid in a 1-h dwell. We also sought to elucidate whether diffusion of radiolabeled [ 18 F]-deoxyglucose in the opposite direction (plasma → dialysate) is also lowered by selective/non-selective GLUT inhibition. Results show that selective GLUT1 inhibition markedly improved UF and enhanced the sodium dip, but no alterations in glucose transport or [ 18 F]-deoxyglucose diffusion could be detected. Non-selective GLUT-inhibition using phloretin showed similar improvements on water and sodium transport, but also markedly decreased diffusion capacity for [ 18 F]-deoxyglucose. We conclude that selective GLUT1 inhibition improved the UF efficiency in terms of mL of water removed per gram glucose absorbed by almost 70% for 1.5% glucose, implicating a role for GLUT1 in glucose mediated osmotic water transport in PD. Selective inhibitors of facilitative glucose transporter 1 may be promising agents to improve UF efficacy in patients treated with PD. Translational Statement Peritoneal dialysis (PD) is limited by systemic and local glucose toxicity. Here we used a highly selective inhibitor of facilitative glucose channel 1 (GLUT1) in a rat model of PD, and show marked, direct improvements in osmotic water removal (UF) per gram glucose absorbed. Inhibitors of GLUT1 may provide marked improvements in UF in patients on PD.

Introduction: In kidney transplantation (KT), the role of the intravascular innate immune system (IIIS) in response to ischemia-reperfusion injury (IRI) is not well-understood. Here, we studied parallel changes in the generation of key activation products of the proteolytic cascade systems of the IIIS following living donor (LD) and deceased donor (DD) transplantation and evaluated potential associations with clinical outcomes. Methods: In a cohort study, 63 patients undergoing LD (n = 26) and DD (n = 37) transplantation were prospectively included. Fifteen DD kidneys were preserved with hypothermic machine perfusion (HMP), and the remaining were cold stored. Activation products of the kallikrein-kinin, coagulation, and complement systems were measured in blood samples obtained systemically at baseline and locally from the transplant renal vein at 1, 10, and 30 minutes after reperfusion. Results: DD kidneys exhibited a prompt and interlinked activation of all 3 cascade systems of IIIS postreperfusion, indicating a robust and local thrombo-inflammatory response to IRI. In this initial response, the complement activation product sC5b-9 exhibited a robust correlation with other IIIS activation markers and displayed a strong association with short-term and mid-term (24-month) graft dysfunction. In contrast, LD kidneys did not exhibit this thrombo-inflammatory response. The use of HMP was associated with reduced thromboinflammation and preserved mid-term kidney function. Conclusion: Kidneys from DD are vulnerable to a prompt thrombo-inflammatory response to IRI, which adversely affects both short-term and long-term allograft function. Strategies aimed at minimizing graft immunogenicity prior to reperfusion are crucial to mitigate the intricate inflammatory response to IRI.

Introduction: Intradialytic hypotension is a common complication of haemodialysis, but uncommon in peritoneal dialysis (PD). This may be due to lower ultrafiltration rates in PD compared to haemodialysis, allowing for sufficient refilling of the blood plasma compartment from the interstitial volume, but the underlying mechanisms are unknown. Here we assessed plasma volume and hemodynamic alterations during experimental PD with high versus low ultrafiltration rates. Methods: Experiments were conducted in two groups of healthy Sprague-Dawley rats: one group with a high ultrafiltration rate (N = 7) induced by 8.5% glucose and a low UF group (N = 6; 1.5% glucose), with an initial assessment of the extracellular fluid volume, followed by 30 min PD with plasma volume measurements at baseline, 5, 10, 15 and 30 min. Mean arterial pressure, central venous pressure and heart rate were continuously monitored during the experiment. Results: No significant changes over time in plasma volume, mean arterial pressure or central venous pressure were detected during the course of the experiments, despite an ultrafiltration (UF) rate of 56 mL/h/kg in the high UF group. In the high UF group, a decrease in extracellular fluid volume of -7 mL (-10.7% (95% confidence interval: -13.8% to -7.6%)) was observed, in line with the average UF volume of 8.0 mL (standard deviation: 0.5 mL). Conclusion: Despite high UF rates, we found that plasma volumes were remarkably preserved in the present experiments, indicating effective refilling of the plasma compartment from interstitial tissues. Further studies should clarify which mechanisms preserve the plasma volume during high UF rates in PD.

Abstract Background and Aims Peritoneal dialysis (PD) is the most common home dialysis treatment, and is associated with lower societal costs and increased patient autonomy. During routine PD, the exact amount of fluid inside the peritoneal cavity is in general unknown, and the peritoneal catheter may also change position, which may lead to a large residual volume, inefficient dialysis, and potentially harmful overfill. A practical, clinical method to estimate the residual volume is from the dilution of a marker molecule before and after fill of a known volume of fresh dialysis fluid. However, ultrafiltration and transport of the marker molecule during the fill phase may introduce large errors, especially when low molecular weight marker molecules are utilized. Method Here we performed a retrospective analysis of clinical data consisting of 56 residual volume estimations utilizing either 1.5% glucose (n = 28) or 4.25% glucose fluid (n = 28). Residual volumes were estimated using creatinine, urea and albumin. The contribution of ultrafiltration and mass transport of the marker molecules were quantified using the Three-pore model. Results During the fill phase, the median ultrafiltration volume was 16 mL (IQR 3 to 31) in 1.5% glucose dwells, compared to 52 mL (IQR 38 to 66) during 4.25% glucose dwells (Fig. 1). Assuming that 40% of the ultrafiltration occurs as free-water transport, the median difference between Three-pore model and albumin dilution equation residual volumes were only -1 mL (-8 to 5.5) in 1.5% glucose dwells and 1 mL (-3 to 6) in 4.25% glucose dwells (Fig. 2A). Dilution-based calculations using creatinine overestimated residual volumes by 121 mL (IQR 103 to 149) during fill of 1.5% glucose fluid and 255 mL (IQR 208 to 323) during 4.25% glucose fluid fill (Figure 2B). Overestimation of a similar magnitude was observed in urea calculated residual volumes (Figure 2B). Comparison of intraperitoneal creatinine and urea mass before and after fill revealed a median creatinine mass increase of 37% (creatinine) and 48% (urea) during the fill phase. Conclusion This analysis highlights the inherent limitations of residual volume estimations using low molecular weight marker molecules, as both ultrafiltration and dilution marker mass transfer influence results. Since mass transfer rates correlate with molecular size, albumin-based dilution equation residual volumes conformed closely with three-pore model residual volumes, while creatinine and urea, which are subject to mass transfer to a greater extent, overestimated residual volumes. Albumin is superior to both creatinine and urea, but the latter markers may provide reasonable residual volume estimates, especially if the purpose is to exclude a large residual volume.