Extraction and Analysis of Interstitial Fluid, and Characterisation of the Interstitial Compartment in Kidney Disease

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Abstract

Kidney failure results in fluid and toxin accumulation within body fluid compartments, contributing to the excess mortality seen in this condition. Such uremic toxins have been measured in plasma, with levels assumed to reflect extraplasmatic concentrations such as in interstitial fluid (ISF). ISF is separated from plasma by nanometre-order microvascular pores; toxins may not circulate “freely” between the two compartments. This work set out to characterise the ISF in uremic subjects, with the hypothesis that there may be differences with plasma. Any such difference may be clinically relevant, owing to the much larger size of the ISF compartment, its proximity to cell metabolic processes, and its expansion in renal impairment.We used a modified microdialysis technique to successfully sample subcorneal ISF of some the uremic toxins (urea, creatinine, urate, phosphate). Reverse iontophoresis (RI) was also used as a non-invasive technique to sample epidermal ISF of urea. Hollow microneedles were developed and their ability to extract ISF tested in CKD patients and controls. The mechanical properties (pressure, volume, permeability) and biochemical composition (proteomic and metabolomic profiles) of the interstitial compartment were also investigated.Microdialysis and RI performed very well as interstitial uremic toxin sampling techniques. Small differences were seen in steady states between ISF and plasma urea, creatinine, phosphate and urate, with slightly lower ISF levels. Dialysis seemed to enhance this difference, with a lag in the clearance of ISF toxins seen in some patients, most remarkable with phosphate. Metabolomic analysis identified several uremic toxins in ISF, whilst proteomics found some significant differences between the two compartments, with toxins like beta-2 microglobulin occurring in ISF only. Microneedle arrays successfully extracted ISF in 68.8% of patients with oedema. Successful extraction of ISF with microneedles occurred mainly in oedematous patients, who were found to have raised interstitial pressures (ISP) and volumes. ISP correlated significantly with body fluid volumes and seemed time-dependent, lower in more chronic oedema. ISP and volumes also correlated with the oedema depitting time (after thumb pressure), a potential novel parameter that probably relates to tissue hydraulic conductivity and hence volume status and fluid mobility within the interstitium.This study demonstrates that interstitial fluid may need to be considered as a separate active compartment in patients with renal dysfunction, with a different “uremic" composition and unique pathophysiological characteristics that cannot be explained by blood compartment based measurements alone. There is a need for more studies, to further characterise this compartment and elucidate its importance.

Layman's Abstract

Patients with kidney disease lose the ability to remove poisonous substances and fluids from their body, causing these to accumulate. The accumulation of these toxins and fluid contributes the high mortality seen with kidney disease. Dialysis aims to remove these toxins but only addresses the blood compartment, which represents less than 7% of body fluids. The assumption, that toxins are equality distributed in body fluid compartments may be wrong, and as such lead to poor removal of toxins by dialysis and their accumulation in compartments such as interstitial or tissue fluid. Tissue fluid composition is unknown in kidney disease patients. This study looked at tissue fluid levels of certain toxins like urea, creatinine, uric acid and phosphate and found them to be different from their blood levels. During dialysis, removal of these toxins from blood was not matched by their removal from tissue fluid, indicating that such toxins may be poorly removed from tissue fluid by dialysis. We measured body fluid volumes in these patients and found that tissue fluid volume was markedly increased, more than 7 times the blood fluid volume in most patients. This means that tissue fluid is a large toxin and fluid reservoir in kidney patients, and that the current blood-based approach to measuring toxins and removing them may be flawed. Extraction of tissue fluid can be quite challenging. We developed microneedles (tiny needles) and showed that these could be used to extract tissue fluid from skin, without causing pain or bleeding. Development of such technology will improve understanding of tissue fluid and perhaps lead to remote monitoring at home, using microneedle patches to extract tissue fluid. We also used a small electric current to successfully extract urea (a toxin that accumulates in kidney failure) from the skin, as a possible screening and monitoring method for kidney disease. This study shows that blood measurements do not necessarily reflect other body fluids (tissue fluid). This implies that blood-based measurements of toxins and their removal by dialysis may be fundamentally flawed. More research on tissue fluid will help to better understand toxicity in kidney failure and maybe help in designing better methods of toxin removal

Keyword(s)

bioimpedance; interstitial fluid; interstitial pressure; kidney disease; metabolomics; microdialysis; microneedles; proteomics; reverse iontophoresis; uremic toxins; volume assessment

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