Study to Examine the Effect of the Diuretic Furosemide on the Plasma Levels of Toxins and the Removal of Toxins from the Blood in Patients with Chronic Kidney Disease

The goal of this observational study is to examine the interaction between the diuretic furosemide and certain toxins called protein-bound uremic toxins (PBUTs) in patients with chronic kidney disease (CKD). The main question it aims to answer is: what is the effect of furosemide on plasma levels of PBUTs in patients with CKD? Besides, the investigators will also look at the effect of furosemide on the excretion of PBUTs via the urine.

Participants will be included in the study once they will be prescribed furosemide as part of routine patient care. Before they start with the furosemide treatment, patients will undergo the following:

* Blood pressure measurement
* Blood sample withdrawal
* Urine sample collection
* 12-hour urine collection

Then, one to four weeks after starting with furosemide treatment, patients will undergo the following:

* Blood pressure measurement
* Blood sample prior to furosemide intake (Tmin)
* Blood sample withdrawal 90 minutes after furosemide intake (Tmax: time at which the highest furosemide plasma level is expected)
* Urine sample collection 60-120 minutes after furosemide intake
* 12-hour urine collection

The investigators expect furosemide to increase plasma levels of PBUTs by decreasing the renal excretion of the toxins.

Protein-bound uremic toxins are known to accumulate in chronic kidney disease (CKD) and are associated with increased morbidity and mortality. It is therefore crucial to maintain the PBUT levels low in this patient group. Furosemide is often prescribed to CKD patients. However, based on preclinical data, furosemide could affect the renal excretion of PBUTs, either by competing for binding to albumin or by competing for the secretory system in the kidney. It is important to examine the effect of furosemide on the excretion and plasma concentration PBUTs as this might have harmful consequences for patients with CKD.

This study aims to examine the effect of furosemide PBUT plasma levels in patients with CKD as well as the renal PBUT excretion. The investigators expect furosemide to increase plasma levels of PBUTs by decreasing the renal excretion of the toxins.

This study is observational and includes invasive measurements; a prospective repeated measures cohort study design will be used in which PBUT plasma concentrations and excretion will be determined before and after the start of furosemide treatment.

The study population consists of patients with CKD stage 3-5 (\<60 mL/min/1.73m2 for at least three months) who have an indication for furosemide treatment as part of routine patient care. Participants will receive furosemide in a dosage prescribed by their nephrologist as part of their routine patient care; this treatment is not changed by the participation of patients in the study.

A power analysis for a paired samples T-Test was done using GPower (version 3.1.9.4). Data regarding PBUT plasma levels from the study of Tang et al. in 2021 was used in order to calculate the sample size; PCS plasma levels before (average ± standard deviation: 13,481 ± 12,642 nM) and after (average ± standard deviation: 23,000 ± 24,900 nM) furosemide intake were chosen, since PCS is one of the main PBUTs of interest. This calculation showed a required sample size of 34 (one-tail, power = 80%, α = 0.05, d = 0.44, correlation between samples 0.5 based on assumption).

Prior to the start of the furosemide treatment, a blood sample (three tubes/11 mL per withdrawal) and a urine sample will be collected from patients enrolled in the study. Participants will also hand in a 12-hour urine collection and their blood pressure will be measured.

After the start of the furosemide treatment (at least one week after the start of the furosemide treatment so that steady state has been reached), two blood samples, one urine sample, and a 24-hour urine collection will be obtained. Participants will be asked to obtain a 12-hour urine collection and to bring this with them on the day the of the visit. During the visit at the University Medical Center Utrecht, participants will first hand in the 12-hour urine collection and their blood pressure will be measured. Furthermore, a blood sample will be taken prior to the furosemide intake (Tmin). Then, they will take their prescribed furosemide at around the same time as they normally take their medication. 60 minutes after the furosemide intake, participants will be asked to empty their bladder and drink two glasses of water. Then, at Tmax, a blood sample will be collected. Tmax is estimated at around 90 minutes after oral intake; therefore, the target time of sample collection is 90 min with an acceptable range between 60-120 minutes after furosemide intake. Furthermore, a urine sample will be collected between 60 and 120 minutes after furosemide intake in order to best examine the effect of furosemide on PBUT excretion. The exact time of furosemide intake and the time of the blood and urine sample collection will be registered.

The main endpoints of this study are PBUT plasma levels before and 1-4 weeks after the start of furosemide treatment (in a steady state).

Baseline characteristics and study parameters will be presented as either a mean with standard deviation or a median with interquartile range for continuous data, or as a percentage for categorical data. Results with a p\<0.05 will be considered statistically significant. Analyses will be done using the statistical software platforms SPSS and R. Missing data will be excluded from the analyses via pairwise deletion.

Participants will only receive furosemide prescribed by their treating nephrologist as part of routine patient care. In addition, three blood sample drawings, two urine samples, and two 12-hour urine collections will be needed. Thus, the risk associated with participation is negligibly low. Participation to the study will include two site visits. These visits will be combined with routine check-ups as much as possible.

Eligibility Criteria:
Inclusion Criteria:

* An age of 18 years or older
* An eGFR \<60 mL/min/1.73m2 for at least three months (diagnosis of CKD stage 3-5)
* An indication for the start of treatment with furosemide as part of routine patient care
* Willingness to participate in the study and a signed informed consent

Exclusion Criteria:

* Patients who are already on furosemide treatment
* Patients with a liver disease with hyperbilirubinemia
* Patients who receive any type of renal replacement therapy (peritoneal dialysis, haemodialysis)
* Patients with end-stage renal failure without residual diuresis
* Patients who will start with medication simultaneously with start of furosemide treatment that might interfere with PBUT excretion or PBUT protein binding
* Patients who are incapacitated