Effect of furosemide on body composition and urinary proteins that mediate tubular sodium and sodium transport—A randomized controlled trial
2020; Wiley; Volume: 8; Issue: 24 Linguagem: Inglês
10.14814/phy2.14653
ISSN2051-817X
AutoresFrank Holden Mose, A. E. Oczachowska-Kulik, Robert A. Fenton, Jesper Nørgaard Bech,
Tópico(s)Thermoregulation and physiological responses
ResumoPhysiological ReportsVolume 8, Issue 24 e14653 ORIGINAL RESEARCHOpen Access Effect of furosemide on body composition and urinary proteins that mediate tubular sodium and sodium transport—A randomized controlled trial Frank Holden Mose, Corresponding Author Frank Holden Mose frchri@rm.dk orcid.org/0000-0002-5830-5814 University Clinic in Nephrology and Hypertension, Department of Medicine, University of Aarhus and Gødstrup Hospital, Holstebro, Denmark Correspondence Frank Holden Mose, MD, PhD, Associate Professor, University Clinic in Nephrology and Hypertension, Department of Medical Research, Holstebro Hospital, Laegaardvej 12, 7500 Holstebro, Denmark. Email: frchri@rm.dkSearch for more papers by this authorAnna Ewa Oczachowska-Kulik, Anna Ewa Oczachowska-Kulik University Clinic in Nephrology and Hypertension, Department of Medicine, University of Aarhus and Gødstrup Hospital, Holstebro, DenmarkSearch for more papers by this authorRobert Andrew Fenton, Robert Andrew Fenton Department of Biomedicine, Aarhus University, Aarhus, DenmarkSearch for more papers by this authorJesper Nørgaard Bech, Jesper Nørgaard Bech University Clinic in Nephrology and Hypertension, Department of Medicine, University of Aarhus and Gødstrup Hospital, Holstebro, DenmarkSearch for more papers by this author Frank Holden Mose, Corresponding Author Frank Holden Mose frchri@rm.dk orcid.org/0000-0002-5830-5814 University Clinic in Nephrology and Hypertension, Department of Medicine, University of Aarhus and Gødstrup Hospital, Holstebro, Denmark Correspondence Frank Holden Mose, MD, PhD, Associate Professor, University Clinic in Nephrology and Hypertension, Department of Medical Research, Holstebro Hospital, Laegaardvej 12, 7500 Holstebro, Denmark. Email: frchri@rm.dkSearch for more papers by this authorAnna Ewa Oczachowska-Kulik, Anna Ewa Oczachowska-Kulik University Clinic in Nephrology and Hypertension, Department of Medicine, University of Aarhus and Gødstrup Hospital, Holstebro, DenmarkSearch for more papers by this authorRobert Andrew Fenton, Robert Andrew Fenton Department of Biomedicine, Aarhus University, Aarhus, DenmarkSearch for more papers by this authorJesper Nørgaard Bech, Jesper Nørgaard Bech University Clinic in Nephrology and Hypertension, Department of Medicine, University of Aarhus and Gødstrup Hospital, Holstebro, DenmarkSearch for more papers by this author First published: 23 December 2020 https://doi.org/10.14814/phy2.14653Citations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Background Furosemide inhibits the sodium potassium chloride cotransporter (NKCC2) in the thick ascending limb of the loop of Henle and increases urinary water and sodium excretion. This study investigates the effect of furosemide on body composition estimated with multifrequency bioimpedance spectroscopy (BIS) technique and urinary proteins from NKCC2. Methods This study is a randomized, placebo-controlled, crossover study where healthy subjects received either placebo or 40 mg furosemide on two separate occasions, where body composition with BIS, renal function, proteins from tubular proteins that mediate sodium and water transport, and plasma concentrations of vasoactive hormones were measured before and after intervention. Results We observed an expected increased diuresis with a subsequent reduction in bodyweight of (−1.51 ± 0.36 kg, p < .001) and extracellular water (ECW; −1.14 ± 0.23 L, p < .001) after furosemide. We found a positive correlation between the decrease in ECW and a decrease in bodyweight and a negative correlation between the decrease in ECW and the increase in urinary output. Intracellular water (ICW) increased (0.47 ± 0.28 L, p < .001). Urinary excretion of NKCC2 increased after furosemide and the increase in NKCC2 correlated with an increase in urine output and a decrease in ECW. Conclusion We found BIS can detect acute changes in body water content but the method may be limited to estimation of ECW. BIS demonstrated that furosemide increases ICW which might be explained by an extracellular sodium loss. Finally, urinary proteins from NKCC2 increases after furosemide with a good correlation with diuresis end the decrease in ECW. 1 BACKGROUND The kidneys regulate fluid and sodium homeostasis which becomes evident in renal failure where fluid retention is often present (Yerram et al., 2010). The mechanisms for fluid retention are not completely described but includes decreased number of nephrons, abnormal activity of tubular cells that regulate water and sodium excretion, abnormal function of vasoactive hormones that regulate fluid and sodium homeostasis including the renin-angiotensin-system and natriuretic peptides and change in central blood volume and blood pressure that lead to change in renal perfusion, activity in vasoactive hormones and sympathetic nervous activity (Raina et al., 2018; Zoccali et al., 2017). Intuitively the kidney must play a central role in the regulation of the different fluid compartments in the body such as intracellular and extracellular volume but this is not well understood (Matthie, 2008; Wabel et al., 2009). Methods using multifrequency bioimpedance spectroscopy (BIS) technique may help to improve our understanding of body composition, fluid regulation, and treatment of volume overload in different stages of renal dysfunction (Arroyo et al., 2015; Ersoy Dursun et al., 2019; Hur et al., 2013; Lukaski et al., 2019; Onofriescu et al., 2014). The activity of tubular proteins that mediate sodium transport is difficult to measure directly but surrogate markers such as protein fragments from transporters have previously been used (Al Therwani et al., 2017; Graffe et al., 2012; Jensen et al., 2014; Pedersen et al., 2001). The effect of furosemide on urinary excretion of proteins from the furosemide sensitive sodium potassium chloride cotransporter (NKCC2) has to our knowledge not been investigated previously (Huang et al., 2016). We therefore hypothesized that furosemide treatment increases urine flow and causes a reduction in bodyweight associated with reductions in ECW and ICW measured with BIS. In addition, the effect of furosemide will change u-NKCC2 reflecting u-NKCC2 activity. Finally, the changes in u-NKCC2 are associated with changes in fluid distribution in the body. We investigated these hypotheses in a study designed as a randomized, placebo-controlled, crossover study where subjects received either placebo or furosemide on two separate occasions, where body composition, renal function, proteins that mediate tubular sodium and water transport, and plasma concentrations of vasoactive hormones were measured. 2 METHODS 2.1 Design The study was a randomized, single-blinded, placebo-controlled, crossover trial (Figure 1). After inclusion subjects were allocated to treatment via computer-generated randomization and received furosemide or 5% glucose (placebo) on examination days in a random order. Examinations were separated by a washout period of at least 2 weeks. FIGURE 1Open in figure viewerPowerPoint Study design Furosemide (Furix, 4 ml of 10 mg/ml, Nycomed Danmark) and isotonic glucose (4 ml 50 g glucosemonohydrate/l, Baxter) were identical in appearance to the study subjects. Furosemide was given at a dose of 40 mg (4 ml) intravenously. Glucose was chosen as placebo to minimize sodium intake. 2.2 Effect variables Extracellular water (ECW) was chosen as the main effect variable. Other effect variables were intracellular water (ICW), ECW/ICW, bodyweight, GFR, plasma sodium (p-Na), serum osmolality, FENa (fractional excretion of sodium), free water clearance (CH2O), urinary excretions of aquaporin-2 (u-AQP2), epithelial sodium channels (u-ENaCγ), sodium chloride cotransporter (u-NCC) and sodium potassium chloride cotransporter (u-NKCC2), urinary osmolality, plasma concentration of vasopressin (p-AVP), renin (PRC), angiotensin II (p-AngII) and aldosterone (p-Aldo), brachial systolic and diastolic blood pressure (DBP, SBP), and heart rate (HR). 2.3 Recruitment Subjects were consecutively recruited by advertisements in local newspapers in the area of Holstebro, Denmark. Written and oral information that included safety concerns of study medication was given, following a written consent. After the written consent was obtained the screening examination was performed. A clinical history was taken and examination was performed, blood was drawn and urine samples were collected. ECG was performed to ensure that the subject fulfilled the inclusion criteria and did not meet the exclusion criteria. Screening examination included physical examination, medical history, clinical biochemistry, urine albumin analysis ECG, and ambulatory BP measurement. 2.4 Subjects Inclusion criteria: Healthy women and men, BMI 18.5–30.0 kg/m2, age 18–45 years, fertile women must use safe anticonception. Exclusion criteria: Clinical signs of or history with diseases in the central nervous system, thyroid gland, heart and lungs, liver or kidneys, malignancies, diabetes mellitus, ambulatory blood pressure >130 mmHg systolic and/or >80 mmHg diastolic, clinical important deviations in screening urine or blood samples, medical or alcohol abuse, smoking, nursing or pregnancy, allergy or intolerance towards furosemide, unwillingness to participate. Withdrawal criteria: Noncompliance or development of exclusion criteria. 2.5 Number of subjects With a significance level of 5% and a power of 80% a total of 22 subjects were needed to detect a 1.25 L difference in ECW (SD 2 L). Because incomplete voiding during examination days was expected in some subjects, it was estimated that 24 subjects should finish the trial. 2.6 Experimental procedure Prior to examinations subjects received a 4-day standard diet, as previously described (Jensen et al., 2013, 2014; Matthesen et al., 2013; Mose et al., 2015). The diet comprised three minor meals and three main meals. The complete diet contained 11,000 kJ/day and was composed of 55% carbohydrates, 30% fat, and 15% protein. The total sodium content was 150 mmol per day. Subjects were instructed to eat variedly from the diet until satiated. Daily fluid intake was recommended to 2.75 L (250 ml per 1,000 kJ). No consumption of alcohol was allowed. Up to two cups of coffee or tea pr. day were allowed. Twenty-four-hour urine collection was performed before each examination. Twenty-four-hour urine was analyzed for sodium, creatinine, albumin, AQP2, ENaCγ, NCC, and NKCC2. After an overnight fast, subjects arrived for examination at 8 a.m. Two indwelling catheters for blood sampling and administration of 51Cr-EDTA and furosemide or glucose (placebo) were placed in cubital veins, one in each arm. Every 30 min after arrival, participants received an oral water load of 175 ml of tap water. Subjects were kept in a supine position in a quiet, temperature-controlled room (22°C–25°C). Standing or sitting was only permitted during voiding. At 11 a.m. injection furosemide or glucose was given according to randomization. Blood and urine samples were collected every 30 min from 9:30 a.m. to 2.30 p.m. Urine collections were analyzed for 51Cr-EDTA, sodium, creatinine, AQP2, ENaCγ, NCC, and NKCC2. The first three clearance periods from 9:30 a.m. to 11 a.m. were used as the baseline period. The baseline period was followed by clearance periods as described above. Blood samples were drawn at 11 a.m. (baseline) just prior to infusion of study medication and again 1 and 2 hr after infusion of study medication for determination of p-AVP, PRC, p-AngII, and p-Aldo. 2.7 Blood pressure measurements Office BP measured during examination was recorded by the semiautomatic, oscillometric device, Omron 705IT (Omron Matsusaka CO. Ltd.). Bioimpedance spectroscopy. Bioimpedance spectroscopy (BIS) was measured using Body Composition Monitor (BCM, Fresenius Medical Care) and was used according to the manufacturer's instructions. Bioimpedance measurements performed at a spectrum of 50 frequencies between 5 and 1,000 kHz allow to differentiate between extra- and intracellular fluid, as low electronic currents only flow through extracellular water because they cannot pass cell membranes (Moissl et al., 2006). Parameters of volume status and body composition are calculated by the BCM using two physiological models: The body volume model is used to calculate ECW, ICW, and total body water (TBW) and the body composition model differentiates normally hydrated fat mass, normally hydrated lean mass and a remaining proportion of water, and lays the foundation to calculate parameters of adipose tissue, lean tissue and the so-called overhydration (OH; Chamney et al., 2007). OH is mainly part of extracellular fluid and reference values for OH lie between −1 and +1 L. 2.8 Biochemical analyses Urine samples were kept frozen at −20°C until assayed. U-AQP2, u-ENaCγ, and U-NKCC2 were measured by radioimmunoassay as previously described (Al Therwani et al., 2017; Graffe et al., 2012; Jensen et al., 2014; Pedersen et al., 2001). Antibodies were raised in rabbits to synthetic peptides for AQP2, ENaCγ, and NKCC2. Urine samples for measurement of NCC were thawed and centrifuged at 2,200 g for 10 min before storage. A sample volume—standardized to osmolality—was freeze-dried and kept at −20°C until analysis. For analysis, the freeze-dried samples were suspended in 200 µl albumin buffer (phosphate 40 mM, albumin 2 g/L) and 100 µl assay buffer. Assay buffer contained 40 mM phosphate, albumin 2 g/L, 0.36% EDTA, and 1 ‰ Triton-X-100. 50 µl of antibody was added to each tube and incubated for 24 hr at 4°C. 50 µl of 125I-NCC was added and incubated for further 24 hr. 100 µl of bovine gamma globulin and 2 ml of polyethylene glycol were added. After 1 hr, the tubes were centrifuged at 4,100 g for 20 min. at 4°C. The supernatant was discarded and the precipitate was counted in a gamma counter. A standard curve was constructed (i.e., 9 points increasing from 0 pg/tube to 4,000 pg/tube) to read of the unknown amounts of NCC in urine extracts. For six consecutive standard curves, the zero standard was 81.3 ± 1.4% binding. For increasing amounts of NCC-standard, the binding inhibition was 79.6 ± 1.3% (31 pg/tube), 77.7 ± 1.3% (62.5 pg/tube), 73.0 ± 1,8% (125 pg/tube), 64.1 ± 1,6% (250 pg/tube), 44.9 ± 2.1% (500 pg/tube), 26.9 ± 1.0% (1,000 pg/tube), 17.5 ± 0.7% (2,000 pg/tube), and 12.1 ± 0.3% (4,000 pg/tube). The minimal detection limit was 62.5 pg/tube (zero binding −2 SD). Average nonspecific binding was 5.7 ± 0.6%. The ID 50 (concentration of standard needed for 50% binding inhibition) was 575 ± 34 pg/tube. The intraassay coefficient of variation was 8.2% (n = 40, 4 assays) and interassay coefficient of variation was 11.1% (n = 36, 9 assays). Iodination was obtained using chloramine T with 40 µ antigen and 37 MBq I125. I125-NCC was separated on a G25 Sephadex column after the process was terminated using 20% human albumin. NCC was obtained from Genscript Biotech (Netherlands). The NCC antibody was produced and specificity was secured. A 18-amino acid peptide, CRRDCPWKISDEEITKNR (the NH2 terminal cysteine added for conjugation) corresponding to amino acids 943–959 of human NCC (accession# AAC50355.1) was produced by standard solid-phase techniques and conjugated to keyhole limpet hemocyanin (KLH) via covalent linkage to the NH2-terminal cysteine (Genscript USA). The antibody was affinity purified (termed #8285) from terminal bleed serum using the immunizing peptide as described previously (Fenton et al., 2007). The antibody #8285 titer was determined to be >1:512,000 using ELISA and NCC peptide-conjugated plates. Antibody #8285 specificity was determined by: (a) western blotting of MDCK cells expressing human NCC, where a strong signal was only observed in transfected cells (Rosenbaek et al., 2017); (b) western blotting of human whole kidney, cortex or medulla tissue, showing a strong band of the characteristic molecular mass of NCC only in cortical samples (c) triple immunohistochemical labeling (as previously described) of mouse kidney using markers of late DCT (CalbindinD28) and connecting tubule/collecting duct (Aquaporin-2; Pedersen et al., 2010); (d) immunohistochemical labeling of tubules morphologically similar to the distal convoluted tubule in human kidney sections. Blood samples collected for measurements of vasoactive hormones were centrifuged and plasma was separated, and kept frozen until assayed as previously described (Al Therwani et al., 2014). AVP and Ang II were extracted from plasma and then determined by radioimmunoassay (Al Therwani et al., 2014; Pedersen et al., 1984, 1993). PRC was determined by immunoradiometric assay as previously described (Al Therwani et al., 2014). Aldo was determined by radioimmunoassay as previously described (Mose et al., 2014). Glomerular filtration rate (GFR) was determined with constant infusion clearance technique with 51Cr-EDTA as a reference substance (Jensen et al., 2014; Mose et al., 2015). Urine and plasma concentration of creatinine, sodium, and albumin were measured by routine methods at the Department of Clinical Biochemistry. 2.9 Calculations In 24-hr urine collections, GFR was estimated by creatinine clearance. Fractional excretions of sodium (FENa) was calculated with the formula: FENa = (UNa * V/CNa)/GFR, where UNa and CNa are urine and plasma concentrations of Na+ and V is urine flow in ml/min. Free water clearance (CH2O) was calculated using the formula: CH2O = UO − Cosm, where UO is urinary output and Cosm is osmolar clearance. Urine concentration of variables is adjusted for urinary flow resulting in an excretion rate and for creatinine excretion giving an approximate adjustment for glomerular filtration. 2.10 Statistics Data are presented as medians with 25% and 75% percentiles in brackets, if normality was not present and as means ± standard deviations (SD), if data showed normality. A paired comparison within and between groups was performed with paired t test or Wilcoxon signed-rank test. A general linear model for repeated measures (GLM) was performed to test the difference in responses to furosemide during the experimental procedure. If normality was not present, data were logarithmic transformed before GLM. Friedman's test was used to test if deviations within the treatment of vasoactive hormones occurred during the experimental procedure. Correlations were performed with Pearson correlation. Statistical significance was defined as p < .05. Statistical analyses were performed using PASW version 20.0.0 (SPSS Inc.). 2.11 Ethics The study was approved by the Danish Health and Medicines Authority (EudraCT number: 2012-003815-71) and the Regional Committee on Biomedical Research Ethics (case number:1-16-02-540-14). It was carried out in accordance with the Declaration of Helsinki and was monitored by the Good Clinical Practice Unit from Aarhus and Aalborg Universities. A signed informed consent form was obtained from each patient. 3 RESULTS 3.1 Demographics Forty subjects were screened for participation and included in the trial. 19 subjects were excluded due to medication use (1), low potassium (1), anemia (1), hypertension (1), elevated alaninaminotransferase (1), smoking (1), heart murmurs (2), abnormal ECG (1), no possible cubital intra-venous access and withdrawal of consent (8). Thus, 21 healthy subjects were included and completed the trial. Twenty-one subjects (13 females, 8 males), had a mean BMI 24.1 ± 2.5 kg/m2, age 26 ± 5 years, ambulatory BP 118/71 ± 8/6 mmHg, p-creatinine 73 ± 10 μmol/L, urine albumin 6 (1;9) mg/L, p-hemoglobin 8.6 ± 0.7 mmol/L. 3.2 Bioimpedance spectroscopy and bodyweight At baseline, ECW, ICW, OH, ECW/ICW, and bodyweight were similar between treatments (Table 1). OH was negative at baseline indicating small dehydration, which was attenuated after furosemide. Furosemide reduced bodyweight (−1.51 ± 0.36 kg, p < .001). The change in ECW and ICW from baseline is shown in Figure 2. The ECW/ICW ratio was reduced after furosemide. TABLE 1. Effect of furosemide on bioimpedance spectroscopy (BIS) and plasma sodium and osmolality Baseline 1 hr post intervention (60 min) 2 hr post intervention (60 min) p-value (difference in response) ECW (L) Placebo 15.9 ± 3.4 15.9 ± 3.4 15.9 ± 3.4 <.001 Furosemide 15.7 ± 3.7 14.9 ± 3.5* 14.5 ± 3.5* ICW (L) Placebo 22.5 ± 6.2 22.6 ± 6.1 22.6 ± 6.0 <.001 Furosemide 22.5 ± 6.4 22.9 ± 6.4* 23.0 ± 6.6* ECW/ICW Placebo 0.71 ± 0.05 0.71 ± 0.04 0.71 ± 0.04 <.001 Furosemide 0.71 ± 0.04 0.66 ± 0.04* 0.64 ± 0.04* Overhydration (L) Placebo −0.7 ± 0.8 −0.7 ± 0.8 −0.8 ± 0.9 <.001 Furosemide −0.9 ± 0.9 −1.8 ± 0.8 −2.1 ± 0.7 Bodyweight (kg) Placebo 70.7 ± 10.8 70.6 ± 10.9* 70.5 ± 10.9* <.001 Furosemide 70.6 ± 10.4 69.4 ± 10.6* 69.1 ± 10.5* P-Sodium (mmol/l) Placebo 139 ± 1 138 ± 1* 138 ± 1* .205 Furosemide 138 ± 1 138 ± 1 137 ± 1* S-Osmolality (mmol/kg) Placebo 285 ± 4 284 ± 4* 282 ± 4* .423 Furosemide 285 ± 5 284 ± 4 281 ± 5* Note Extracellular volume (ECW), intracellular volume (ICW), bodyweight, p-sodium, and s-osmolality were measured before furosemide or placebo infusion and repeated 1 and 2 hr after infusion. Data are shown as means ± SD. p-value represents the probability of difference in response to furosemide (response from baseline to 1 hr after injection) between treatments. Students t test was used to test the difference in response to injection between treatments and to test statistically significant difference from baseline, *p < .05. FIGURE 2Open in figure viewerPowerPoint Change in ECW (a) and ICW (b) 60 and 120 min after furosemide and placebo infusion. Statistically significant difference from baseline: * = p < .05 3.3 Plasma sodium and serum osmolality P-sodium and u-osmolality decreased after both placebo and furosemide and to a similar extent (Table 1). 3.4 GFR and tubular function during baseline conditions The volume and composition of 24-hr urinary collection made prior to the two examinations were not significantly different between treatments (Table 2). Similarly, at baseline during examinations, no difference in evaluated parameters was found (Table 3), GFR, urine output, CH2O, FEJNa, U-AQP-2, u-ENaCγ, u-NCC, and u-NKCC2 were similar between treatment arms (Tables 3 and 4). TABLE 2. 24-hr urine collection prior to two examinations Placebo Furosemide p-value Urine output (ml/minute) 1.68 ± 0.61 1.68 ± 0.47 1.000 CH2O (ml/minute) −0.21 ± 0,54 −0.30 ± 0.57 .578 U-creatinine (mmol/24 hr) 14.7 ± 4.4 14.8 ± 3.6 .950 Creatinine clearance (mmol/mL pr. m2) 137 ± 29 139 ± 25 .772 U-Na (mmol/24 hr) 117 ± 49 111 ± 27 .570 FENa (%) 20.1 ± 13 23 ± 18 .387 UAER (mg/24 hr) 5 (4;12) 7 (5;12) .938 U-AQP−2/min (ng/minute) 1.12 ± 0.32 1.12 ± 0.29 .187 U-AQP−2/creatinine (ng/mmol) 111 ± 15 113 ± 29 .684 U-ENaCγ/min (ng/minute) 0.74 ± 0.29 0.73 ± 0.28 .429 U-ENaCγ/creatinine (ng/mmol) 73 ± 21 71 ± 16 .564 U-NCC/min (ng/min) 0.58 ± 0.21 0.63 ± 0.14 .104 U-NCC/creatinine (ng/mmol) 58 ± 15 64 ± 16 .129 U-NKCC2/min (ng/min) 0.86 ± 0.29 0.90 ± 0.25 .402 U-NKCC2/creatinine (ng/mmol) 88 ± 28 90 ± 25 .319 Note Urine output, free water clearance (CH2O), urine excretion of sodium (U-Na) fractional excretion of sodium (FENa) creatinine clearance, urinary excretions rates of albumin (UAER), aquaporin-2 (u-AQP-2/min), γ-fraction of the epithelial sodium channel (u-ENaCγ/min), sodium chloride cotransporter (u-NCC/min), and sodium potassium chloride cotransporter (u-NKCC2/min) and in relation to creatinine (u-AQP-2/creatinine, u-ENaCγ/creatinine, u-NCC/creatinine, u-NKCC2/creatinine. Urine collected from 07.00 a.m. the day before the examination day to 07.00 a.m. on the examination day. Data are shown as means ± SD. Statistics are performed with paired t test or Wilcoxon signed-rank test. TABLE 3. Effect of furosemide on GFR and tubular function Baseline 0–30 min 30–60 min 60–90 min 90–120 min p (GLM within) GFR (51Cr-EDTA clearance) Placebo 97 ± 10 96 ± 11 99 ± 11 97 ± 15 100 ± 12 <.001 Furosemide 96 ± 10 92 ± 12* 87 ± 10* 82 ± 11* 86 ± 12* p (GLM between) .815 Urine output (mL/min) Placebo 7.3 ± 1.4 4.1 ± 1.1* 6.5 ± 1.3 4.7 ± 1.8* 7.3 ± 1.8 <.001 Furosemide 7.2 ± 1.3 24.3 ± 4.4* 20.8 ± 4.0* 12.2 ± 3.6* 8.0 ± 2.8 p (GLM between) <.001 CH2O (ml/min) Placebo 5.0 ± 1.1 1.3 ± 1.2* 3.6 ± 1.2* 2.1 ± 1.4* 3.9 ± 1.4* <.001 Furosemide 5.0 ± 1.3 3.6 ± 1.8* 3.2 ± 1.3* 1.2 ± 1.2* 1.3 ± 1.4* p (GLM between) .164 U-Na (mmol/l) Placebo 25 ± 10 48 ± 20* 33 ± 13* 44 ± 13* 37 ± 10* <.001 Furosemide 23 ± 6 109 ± 7* 107 ± 6* 108 ± 11* 91 ± 20* p (GLM between) <.001 FENa (%) Placebo 0.92 ± 0.44 1.00 ± 0.43* 1.10 ± 0.42* 1.09 ± 0.42* 1.21 ± 0.39* <.001 Furosemide 0.85 ± 0.32 13.08 ± 2.11* 12.43 ± 1.78* 7.27 ± 1.51* 3.93 ± 1.38* p (GLM between) <.001 Note Glomerular filtration rate (GFR), urine output, free water clearance (CH2O) and fractional excretion of sodium (FENa). Urine was collected every 30 min. Data from three baseline periods are pooled and shown as one period. Data are presented as means ± SD. Statistics are performed with a general linear model (GLM) or paired t test. Statistically significant difference from baseline: * = p < .05. TABLE 4. Effect of furosemide on excretion of proteins from aquaporin-2 channels and tubular channels that mediate sodium transport Baseline 0–30 min 30–60 min 60–90 min 90–120 min p (GLM within) U-AQP2 (ng/minute) Placebo 1.19 (0.97;1.41) 1.20 (1.03;1.39) 1.19 (1.01;1.39) 1.09 (0.95;1.27)* 1.31 (1.03;1.43) <.001 Furosemide 1.21 (1.04;1.39) 3.42 (2.83;3.81)* 2.78 (2.18;3.81)* 1.95 (1.55;2.76)* 1.68 (1.30;2.01)* p (GLM between) <.001 U-AQP2/creatinine (ng/mmol) Placebo 115 (104;136) 211 (153;249)* 134 (111;155) 179 (140;132)* 118 (105;143) .001 Furosemide 123 (101;135) 105 (85;129) 100 (73;145) 112 (83;179) 139 (91;205) p (GLM between) .059 U-ENaCγ (ng/minute) Placebo 0.66 (0.52;0.80) 0.63 (0.45;0.83) 0.57 (0.45;0.80) 0.59 (0.42;0.66) 0.65 (0.44;0.77) .017 Furosemide 0.57 (0.48;0.84) 0.76 (0.53;0.89) 0.55 (0.45;0.81) 0.74 (0.62;0.84)* 0.77 (0.64;0.94)* p (GLM between) .355 U-ENaCγ/creatinine (ng/mmol) Placebo 61 (54;70) 105 (87;139)* 63 (53;83) 80 (66;116)* 57 (52;80) <.001 Furosemide 64 (56;71) 19 (14;28)* 18 (15;29)* 47 (37;60)* 64 (55;101) p (GLM between) <.001 U-NCC (ng/min) Placebo 0.58 (0.51;0.77) 0.58 (0.46;0.65)* 0.58 (0.53;0.64) 0.53 (0.44;0.66) 0.59 (0.50;0.67) <.001 Furosemide 0.57 (0.52;0.71) 1.53 (1.35;1.97)* 1.36 (1.24;1.67)* 1.02 (0.83;1.23)* 0.69 (0.65;0.93)* p (GLM between) <.001 U-NCC/creatinine (ng/mmol) Placebo 65 (57;74) 91 (72;112)* 69 (56;79) 82 (61;103)* 57 (47;73) .003 Furosemide 60 (49;68) 54 (37;68)* 46 (38;65) 69 (46;84) 71 (54;95) p (GLM between) .007 U-NKCC2 (ng/min) Placebo 0.96 (0.78;1.14) 0.94 (0.75;1.03)* 0.93 (0.78;1.07) 0.87 (0.69;1.03)* 0.97 (0.70;1.17) <.001 Furosemide 1.00 (0.75;1.11) 4.50 (3.73;5.02)* 3.51 (2.87;4.23)* 2.41 (2.03;2.59)* 1.65 (1.39;2.28)* p (GLM between) <.001 U-NKCC2/creatinine (ng/mmol) Placebo 84 (75;118) 160 (106;197)* 111 (82;118) 122 (92;156)* 92 (68;112)* <.001 Furosemide 91 (69;104) 111 (93;169)* 106 (96;157)* 155 (124;179)* 155 (132;197)* p (GLM between) .245 Note Aquaporin-2 (u-AQP-2/min), γ-fraction of the epithelial sodium channel (u-ENaCγ/min), sodium chloride cotransporter (u-NCC/min), and sodium potassium chloride cotransporter (u-NKCC2/min) and in relation to creatinine (u-AQP-2/creatinine, u-ENaCγ/creatinine, u-NCC/creatinine, u-NKCC2/creatinine. Urine was collected every 30 min. Data from three baseline periods are pooled and shown as one period. Data are shown as medians with 25 and 75 percentiles in brackets p-value represents the probability of difference in response to hypertonic saline (response from baseline to hypertonic saline) between treatments Statistics are performed with a general linear model (GLM), or Wilcoxon signed-rank test. Data were logarithmic transformed before GLM was performed. Statistically significant difference from baseline: * = p < .05. 3.5 GFR and tubular function after furosemide Table 3 shows the effect furosemide on GFR, urine output (UO), CH2O, and FENa. Urine output and FENa increased markedly after furosemide while GFR decreased. Total diuresis was 1959 ml after furosemide and 678 ml after placebo with a total difference of 1,281 ml (p < .001). CH2O decreased after both placebo and furosemide but CH2O had a different response pattern after placebo and furosemide. The initial decrease in CH2O after placebo was attenuated after furosemide, but was exaggerated in the last two clearance periods. 3.6 Urinary excretion of proteins from ENaCγ, AQP2, NCC, and NKCC2 Table 4 shows the furosemide-induced changes in u-AQP2, u-ENaCγ, u-NCC, and u-NKCC2. Different responses in u-AQP2, u-ENaCγ, u-NCC, and u-NKCC2 were observed after placebo and furosemide. U-AQP2, u-ENaCγ, u-NCC, and u-NKCC2 were mainly unchanged after placebo. Small differences were seen in one or two clearance periods with-out a clear pattern for any of the proteins. The only exception is the creatinine adjusted u-NKCC2 excretion where three of four clearance pe
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